Interferon-like proteins and uses thereof

ABSTRACT

The present invention provides Interferon-Like (IFN-L) polypeptides and nucleic acid molecules encoding the same. The invention also provides selective binding agents, vectors, host cells, and methods for producing IFN-L polypeptides. The invention further provides pharmaceutical compositions and methods for the diagnosis, treatment, amelioration, and/or prevention of diseases, disorders, and conditions associated with IFN-L polypeptides.

This application is a divisional of U.S. application Ser. No. 11/200,389filed Aug. 8, 2005, which is a continuation of U.S. application Ser. No.09/927,850, filed Aug. 10, 2001, now abandoned, which is a divisional ofU.S. application Ser. No. 09/724,860 filed Nov. 28, 2000, now abandoned,which claims the benefit of U.S. Provisional Application No. 60/169,720filed Dec. 8, 1999, now abandoned, the disclosure of each of which isexplicitly incorporated by reference herein.

REFERENCE TO THE SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledA-632-US-DIV3.txt, created Feb. 13, 2012, which is 101 KB in size. Theinformation in the electronic format of the Sequence Listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to Interferon-Like (IFN-L) polypeptidesand nucleic acid molecules encoding the same. The invention also relatesto selective binding agents, vectors, host cells, and methods forproducing IFN-L polypeptides. The invention further relates topharmaceutical compositions and methods for the diagnosis, treatment,amelioration, and/or prevention of diseases, disorders, and conditionsassociated with IFN-L polypeptides.

BACKGROUND OF THE INVENTION

Technical advances in the identification, cloning, expression, andmanipulation of nucleic acid molecules and the deciphering of the humangenome have greatly accelerated the discovery of novel therapeutics.Rapid nucleic acid sequencing techniques can now generate sequenceinformation at unprecedented rates and, coupled with computationalanalyses, allow the assembly of overlapping sequences into partial andentire genomes and the identification of polypeptide-encoding regions. Acomparison of a predicted amino acid sequence against a databasecompilation of known amino acid sequences allows one to determine theextent of homology to previously identified sequences and/or structurallandmarks. The cloning and expression of a polypeptide-encoding regionof a nucleic acid molecule provides a polypeptide product for structuraland functional analyses. The manipulation of nucleic acid molecules andencoded polypeptides may confer advantageous properties on a product foruse as a therapeutic.

In spite of the significant technical advances in genome research overthe past decade, the potential for the development of novel therapeuticsbased on the human genome is still largely unrealized. Many genesencoding potentially beneficial polypeptide therapeutics or thoseencoding polypeptides, which may act as “targets” for therapeuticmolecules, have still not been identified.

Accordingly, it is an object of the invention to identify novelpolypeptides, and nucleic acid molecules encoding the same, which havediagnostic or therapeutic benefit.

SUMMARY OF THE INVENTION

The present invention relates to novel IFN-L nucleic acid molecules andencoded polypeptides.

The invention provides for an isolated nucleic acid molecule comprisinga nucleotide sequence selected from the group consisting of:

(a) the nucleotide sequence as set forth in either SEQ ID NO: 1 or SEQID NO: 4;

(b) the nucleotide sequence of the DNA insert in ATCC Deposit No.PTA-976;

(c) a nucleotide sequence encoding the polypeptide as set forth ineither SEQ ID NO: 2 or SEQ ID NO: 5;

(d) a nucleotide sequence which hybridizes under moderately or highlystringent conditions to the complement of any of (a)-(c); and

(e) a nucleotide sequence complementary to any of (a)-(c).

The invention also provides for an isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence encoding a polypeptide which is at least about70 percent identical to the polypeptide as set forth in either SEQ IDNO: 2 or SEQ ID NO: 5, wherein the encoded polypeptide has an activityof the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5;

(b) a nucleotide sequence encoding an allelic variant or splice variantof the nucleotide sequence as set forth in either SEQ ID NO: 1 or SEQ IDNO: 4, the nucleotide sequence of the DNA insert in ATCC Deposit No.PTA-976, or (a);

(c) a region of the nucleotide sequence of either SEQ ID NO: 1 or SEQ IDNO: 4, the DNA insert in ATCC Deposit No. PTA-976, (a), or (b) encodinga polypeptide fragment of at least about 25 amino acid residues, whereinthe polypeptide fragment has an activity of the encoded polypeptide asset forth in either SEQ ID NO: 2 or SEQ ID NO: 5, or is antigenic;

(d) a region of the nucleotide sequence of either SEQ ID NO: 1 or SEQ IDNO: 4, the DNA insert in ATCC Deposit No. PTA-976, or any of (a)-(c)comprising a fragment of at least about 16 nucleotides;

(e) a nucleotide sequence which hybridizes under moderately or highlystringent conditions to the complement of any of (a)-(d); and

(f) a nucleotide sequence complementary to any of (a)-(d).

The invention further provides for an isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence encoding a polypeptide as set forth in eitherSEQ ID NO: 2 or SEQ ID NO: 5 with at least one conservative amino acidsubstitution, wherein the encoded polypeptide has an activity of thepolypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5;

(b) a nucleotide sequence encoding a polypeptide as set forth in eitherSEQ ID NO: 2 or SEQ ID NO: 5 with at least one amino acid insertion,wherein the encoded polypeptide has an activity of the polypeptide setforth in either SEQ ID NO: 2 or SEQ ID NO: 5;

(c) a nucleotide sequence encoding a polypeptide as set forth in eitherSEQ ID NO: 2 or SEQ ID NO: 5 with at least one amino acid deletion,wherein the encoded polypeptide has an activity of the polypeptide setforth in either SEQ ID NO: 2 or SEQ ID NO: 5;

(d) a nucleotide sequence encoding a polypeptide as set forth in eitherSEQ ID NO: 2 or SEQ ID NO: 5 which has a C- and/or N-terminaltruncation, wherein the encoded polypeptide has an activity of thepolypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5;

(e) a nucleotide sequence encoding a polypeptide as set forth in eitherSEQ ID NO: 2 or SEQ ID NO: 5 with at least one modification selectedfrom the group consisting of amino acid substitutions, amino acidinsertions, amino acid deletions, C-terminal truncation, and N-terminaltruncation, wherein the encoded polypeptide has an activity of thepolypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5;

(f) a nucleotide sequence of any of (a)-(e) comprising a fragment of atleast about 16 nucleotides;

(g) a nucleotide sequence which hybridizes under moderately or highlystringent conditions to the complement of any of (a)-(f); and

(h) a nucleotide sequence complementary to any of (a)-(e).

The present invention provides for an isolated polypeptide comprising anamino acid sequence selected from the group consisting of:

(a) the amino acid sequence as set forth in either SEQ ID NO: 2 or SEQID NO: 5; and

(b) the amino acid sequence encoded by the DNA insert in ATCC DepositNo. PTA-976.

The invention also provides for an isolated polypeptide comprising theamino acid sequence selected from the group consisting of:

(a) the amino acid sequence as set forth in either SEQ ID NO: 3 or SEQID NO: 6, optionally further comprising an amino-terminal methionine;

(b) an amino acid sequence for an ortholog of either SEQ ID NO: 2 or SEQID NO: 5;

(c) an amino acid sequence which is at least about 70 percent identicalto the amino acid sequence of either SEQ ID NO: 2 or SEQ ID NO: 5,wherein the polypeptide has an activity of the polypeptide set forth ineither SEQ ID NO: 2 or SEQ ID NO: 5;

(d) a fragment of the amino acid sequence set forth in either SEQ ID NO:2 or SEQ ID NO: 5 comprising at least about 25 amino acid residues,wherein the fragment has an activity of the polypeptide set forth ineither SEQ ID NO: 2 or SEQ ID NO: 5, or is antigenic; and

(e) an amino acid sequence for an allelic variant or splice variant ofthe amino acid sequence as set forth in either SEQ ID NO: 2 or SEQ IDNO: 5, the amino acid sequence encoded by the DNA insert in ATCC DepositNo. PTA-976, or any of (a)-(c).

The invention further provides for an isolated polypeptide comprisingthe amino acid sequence selected from the group consisting of:

(a) the amino acid sequence as set forth in either SEQ ID NO: 2 or SEQID NO: 5 with at least one conservative amino acid substitution, whereinthe polypeptide has an activity of the polypeptide set forth in eitherSEQ ID NO: 2 or SEQ ID NO: 5;

(b) the amino acid sequence as set forth in either SEQ ID NO: 2 or SEQID NO: 5 with at least one amino acid insertion, wherein the polypeptidehas an activity of the polypeptide set forth in either SEQ ID NO: 2 orSEQ ID NO: 5;

(c) the amino acid sequence as set forth in either SEQ ID NO: 2 or SEQID NO: 5 with at least one amino acid deletion, wherein the polypeptidehas an activity of the polypeptide set forth in either SEQ ID NO: 2 orSEQ ID NO: 5;

(d) the amino acid sequence as set forth in either SEQ ID NO: 2 or SEQID NO: 5 which has a C- and/or N-terminal truncation, wherein thepolypeptide has an activity of the polypeptide set forth in either SEQID NO: 2 or SEQ ID NO: 5; and

(e) the amino acid sequence as set forth in either SEQ ID NO: 2 or SEQID NO: 5 with at least one modification selected from the groupconsisting of amino acid substitutions, amino acid insertions, aminoacid deletions, C-terminal truncation, and N-terminal truncation,wherein the polypeptide has an activity of the polypeptide set forth ineither SEQ ID NO: 2 or SEQ ID NO: 5.

Also provided are fusion polypeptides comprising IFN-L amino acidsequences.

The present invention also provides for an expression vector comprisingthe isolated nucleic acid molecules as set forth herein, recombinanthost cells comprising the recombinant nucleic acid molecules as setforth herein, and a method of producing an IFN-L polypeptide comprisingculturing the host cells and optionally isolating the polypeptide soproduced.

A transgenic non-human animal comprising a nucleic acid moleculeencoding an IFN-L polypeptide is also encompassed by the invention. TheIFN-L nucleic acid molecules are introduced into the animal in a mannerthat allows expression and increased levels of an IFN-L polypeptide,which may include increased circulating levels. Alternatively, the IFN-Lnucleic acid molecules are introduced into the animal in a manner thatprevents expression of endogenous IFN-L polypeptide (i.e., generates atransgenic animal possessing an IFN-L polypeptide gene knockout). Thetransgenic non-human animal is preferably a mammal, and more preferablya rodent, such as a rat or a mouse.

Also provided are derivatives of the IFN-L polypeptides of the presentinvention.

Additionally provided are selective binding agents such as antibodiesand peptides capable of specifically binding the IFN-L polypeptides ofthe invention. Such antibodies and peptides may be agonistic orantagonistic.

Pharmaceutical compositions comprising the nucleotides, polypeptides, orselective binding agents of the invention and one or morepharmaceutically acceptable formulation agents are also encompassed bythe invention. The pharmaceutical compositions are used to providetherapeutically effective amounts of the nucleotides or polypeptides ofthe present invention. The invention is also directed to methods ofusing the polypeptides, nucleic acid molecules, and selective bindingagents.

The IFN-L polypeptides and nucleic acid molecules of the presentinvention may be used to treat, prevent, ameliorate, and/or detectdiseases and disorders, including those recited herein.

The present invention also provides a method of assaying test moleculesto identify a test molecule that binds to an IFN-L polypeptide. Themethod comprises contacting an IFN-L polypeptide with a test molecule todetermine the extent of binding of the test molecule to the polypeptide.The method further comprises determining whether such test molecules areagonists or antagonists of an IFN-L polypeptide. The present inventionfurther provides a method of testing the impact of molecules on theexpression of IFN-L polypeptide or on the activity of IFN-L polypeptide.

Methods of regulating expression and modulating (i.e., increasing ordecreasing) levels of an IFN-L polypeptide are also encompassed by theinvention. One method comprises administering to an animal a nucleicacid molecule encoding an IFN-L polypeptide. In another method, anucleic acid molecule comprising elements that regulate or modulate theexpression of an IFN-L polypeptide may be administered. Examples ofthese methods include gene therapy, cell therapy, and anti-sense therapyas further described herein.

In another aspect of the present invention, the IFN-L polypeptides maybe used for identifying receptors thereof (“IFN-L polypeptidereceptors”). Various forms of “expression cloning” have been extensivelyused to clone receptors for protein ligands. See, e.g., Simonsen andLodish, 1994, Trends Pharmacol. Sci. 15:437-41 and Tartaglia et al.,1995, Cell 83:1263-71. The isolation of an IFN-L polypeptide receptor isuseful for identifying or developing novel agonists and antagonists ofthe IFN-L polypeptide signaling pathway. Such agonists and antagonistsinclude soluble IFN-L polypeptide receptors, anti-IFN-L polypeptidereceptor-selective binding agents (such as antibodies and derivativesthereof), small molecules, and antisense oligonucleotides, any of whichcan be used for treating one or more disease or disorder, includingthose disclosed herein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B illustrate the nucleotide sequence of the rat IFN-L gene(SEQ ID NO: 1) and the deduced amino acid sequence of rat IFN-Lpolypeptide (SEQ ID NO: 2). The predicted signal peptide is indicated(underlined);

FIGS. 2A-2B illustrate the nucleotide sequence of the human IFN-L gene(SEQ ID NO: 4) and the deduced amino acid sequence of human IFN-Lpolypeptide (SEQ ID NO: 5). The predicted signal peptide is indicated(underlined);

FIG. 3 illustrates the amino acid sequence alignment of human IFN-Lpolypeptide (huIFN-L; SEQ ID NO: 5), human IFN-β (huIFN-β; SEQ ID NO:7), rat IFN-L polypeptide (raIFN-L; SEQ ID NO: 2), and those amino acidpositions which share some similarity (cons);

FIG. 4 illustrates the nucleotide sequence of the Nde I-Bam HI pAMG21insert (SEQ ID NO: 8) of Amgen strain #3729 and the predicted amino acidsequence (SEQ ID NO: 9) encoded by this insert;

FIG. 5 illustrates the nucleotide sequence of the Nde I-Bam HI pAMG21insert (SEQ ID NO: 10) of Amgen strain #3858 and the predicted aminoacid sequence (SEQ ID NO: 11) encoded by this insert;

FIG. 6 illustrates the nucleotide sequence of the Xba I-Bam HI pAMG21insert (SEQ ID NO: 12) of Amgen strain #4047 and the predicted aminoacid sequence (SEQ ID NO: 13) encoded by this insert;

FIG. 7 illustrates the nucleotide sequence of the Xba I-Bam HI pAMG21insert (SEQ ID NO: 14) of Amgen strain #3969 and the predicted aminoacid sequence (SEQ ID NO: 15) encoded by this insert;

FIG. 8 illustrates the nucleotide sequence of the Nde I-Bam HI pAMG21insert (SEQ ID NO: 16) of Amgen strain #4182 and the predicted aminoacid sequence (SEQ ID NO: 17) encoded by this insert.

DETAILED DESCRIPTION OF THE INVENTION

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All references cited in this application are expressly incorporated byreference herein.

Definitions

The terms “IFN-L gene” or “IFN-L nucleic acid molecule” or “IFN-Lpolynucleotide” refer to a nucleic acid molecule comprising orconsisting of a nucleotide sequence as set forth in either SEQ ID NO: 1or SEQ ID NO: 4, a nucleotide sequence encoding the polypeptide as setforth in either SEQ ID NO: 2 or SEQ ID NO: 5, a nucleotide sequence ofthe DNA insert in ATCC Deposit No. PTA-976, and nucleic acid moleculesas defined herein.

The term “IFN-L polypeptide allelic variant” refers to one of severalpossible naturally occurring alternate forms of a gene occupying a givenlocus on a chromosome of an organism or a population of organisms.

The term “IFN-L polypeptide splice variant” refers to a nucleic acidmolecule, usually RNA, which is generated by alternative processing ofintron sequences in an RNA transcript of IFN-L polypeptide amino acidsequence as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5.

The term “isolated nucleic acid molecule” refers to a nucleic acidmolecule of the invention that (1) has been separated from at leastabout 50 percent of proteins, lipids, carbohydrates, or other materialswith which it is naturally found when total nucleic acid is isolatedfrom the source cells, (2) is not linked to all or a portion of apolynucleotide to which the “isolated nucleic acid molecule” is linkedin nature, (3) is operably linked to a polynucleotide which it is notlinked to in nature, or (4) does not occur in nature as part of a largerpolynucleotide sequence. Preferably, the isolated nucleic acid moleculeof the present invention is substantially free from any othercontaminating nucleic acid molecule(s) or other contaminants that arefound in its natural environment that would interfere with its use inpolypeptide production or its therapeutic, diagnostic, prophylactic orresearch use.

The term “nucleic acid sequence” or “nucleic acid molecule” refers to aDNA or RNA sequence. The term encompasses molecules formed from any ofthe known base analogs of DNA and RNA such as, but not limited to4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinyl-cytosine,pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil,5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,5-carboxy-methylaminomethyluracil, dihydrouracil, inosine,N6-iso-pentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonyl-methyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

The term “vector” is used to refer to any molecule (e.g., nucleic acid,plasmid, or virus) used to transfer coding information to a host cell.

The term “expression vector” refers to a vector that is suitable fortransformation of a host cell and contains nucleic acid sequences thatdirect and/or control the expression of inserted heterologous nucleicacid sequences. Expression includes, but is not limited to, processessuch as transcription, translation, and RNA splicing, if introns arepresent.

The term “operably linked” is used herein to refer to an arrangement offlanking sequences wherein the flanking sequences so described areconfigured or assembled so as to perform their usual function. Thus, aflanking sequence operably linked to a coding sequence may be capable ofeffecting the replication, transcription and/or translation of thecoding sequence. For example, a coding sequence is operably linked to apromoter when the promoter is capable of directing transcription of thatcoding sequence. A flanking sequence need not be contiguous with thecoding sequence, so long as it functions correctly. Thus, for example,intervening untranslated yet transcribed sequences can be presentbetween a promoter sequence and the coding sequence and the promotersequence can still be considered “operably linked” to the codingsequence.

The term “host cell” is used to refer to a cell which has beentransformed, or is capable of being transformed with a nucleic acidsequence and then of expressing a selected gene of interest. The termincludes the progeny of the parent cell, whether or not the progeny isidentical in morphology or in genetic make-up to the original parent, solong as the selected gene is present.

The term “IFN-L polypeptide” refers to a polypeptide comprising theamino acid sequence of either SEQ ID NO: 2 or SEQ ID NO: 5 and relatedpolypeptides. Related polypeptides include IFN-L polypeptide fragments,IFN-L polypeptide orthologs, IFN-L polypeptide variants, and IFN-Lpolypeptide derivatives, which possess at least one activity of thepolypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5. IFN-Lpolypeptides may be mature polypeptides, as defined herein, and may ormay not have an amino-terminal methionine residue, depending on themethod by which they are prepared.

The term “IFN-L polypeptide fragment” refers to a polypeptide thatcomprises a truncation at the amino-terminus (with or without a leadersequence) and/or a truncation at the carboxyl-terminus of thepolypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5. Theterm “IFN-L polypeptide fragment” also refers to amino-terminal and/orcarboxyl-terminal truncations of IFN-L polypeptide orthologs, IFN-Lpolypeptide derivatives, or IFN-L polypeptide variants, or toamino-terminal and/or carboxyl-terminal truncations of the polypeptidesencoded by IFN-L polypeptide allelic variants or IFN-L polypeptidesplice variants. IFN-L polypeptide fragments may result from alternativeRNA splicing or from in vivo protease activity. Membrane-bound forms ofan IFN-L polypeptide are also contemplated by the present invention. Inpreferred embodiments, truncations and/or deletions comprise about 10amino acids, or about 20 amino acids, or about 50 amino acids, or about75 amino acids, or about 100 amino acids, or more than about 100 aminoacids. The polypeptide fragments so produced will comprise about 25contiguous amino acids, or about 50 amino acids, or about 75 aminoacids, or about 100 amino acids, or about 150 amino acids, or about 200amino acids. Such IFN-L polypeptide fragments may optionally comprise anamino-terminal methionine residue. It will be appreciated that suchfragments can be used, for example, to generate antibodies to IFN-Lpolypeptides.

The term “IFN-L polypeptide ortholog” refers to a polypeptide fromanother species that corresponds to IFN-L polypeptide amino acidsequence as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5. Forexample, mouse and human IFN-L polypeptides are considered orthologs ofeach other.

The term “IFN-L polypeptide variants” refers to IFN-L polypeptidescomprising amino acid sequences having one or more amino acid sequencesubstitutions, deletions (such as internal deletions and/or IFN-Lpolypeptide fragments), and/or additions (such as internal additionsand/or IFN-L fusion polypeptides) as compared to the IFN-L polypeptideamino acid sequence set forth in either SEQ ID NO: 2 or SEQ ID NO: 5(with or without a leader sequence). Variants may be naturally occurring(e.g., IFN-L polypeptide allelic variants, IFN-L polypeptide orthologs,and IFN-L polypeptide splice variants) or artificially constructed. SuchIFN-L polypeptide variants may be prepared from the correspondingnucleic acid molecules having a DNA sequence that varies accordinglyfrom the DNA sequence as set forth in either SEQ ID NO: 1 or SEQ ID NO:4. In preferred embodiments, the variants have from 1 to 3, or from 1 to5, or from 1 to 10, or from 1 to 15, or from 1 to 20, or from 1 to 25,or from 1 to 50, or from 1 to 75, or from 1 to 100, or more than 100amino acid substitutions, insertions, additions and/or deletions,wherein the substitutions may be conservative, or non-conservative, orany combination thereof.

The term “IFN-L polypeptide derivatives” refers to the polypeptide asset forth in either SEQ ID NO: 2 or SEQ ID NO: 5, IFN-L polypeptidefragments, IFN-L polypeptide orthologs, or IFN-L polypeptide variants,as defined herein, that have been chemically modified. The term “IFN-Lpolypeptide derivatives” also refers to the polypeptides encoded byIFN-L polypeptide allelic variants or IFN-L polypeptide splice variants,as defined herein, that have been chemically modified.

The term “mature IFN-L polypeptide” refers to an IFN-L polypeptidelacking a leader sequence. A mature IFN-L polypeptide may also includeother modifications such as proteolytic processing of the amino-terminus(with or without a leader sequence) and/or the carboxyl-terminus,cleavage of a smaller polypeptide from a larger precursor, N-linkedand/or O-linked glycosylation, and the like. Exemplary mature IFN-Lpolypeptides are depicted by the amino acid sequences of SEQ ID NO: 3and SEQ ID NO: 6.

The term “IFN-L fusion polypeptide” refers to a fusion of one or moreamino acids (such as a heterologous protein or peptide) at the amino- orcarboxyl-terminus of the polypeptide as set forth in either SEQ ID NO: 2or SEQ ID NO: 5, IFN-L polypeptide fragments, IFN-L polypeptideorthologs, IFN-L polypeptide variants, or IFN-L derivatives, as definedherein. The term “IFN-L fusion polypeptide” also refers to a fusion ofone or more amino acids at the amino- or carboxyl-terminus of thepolypeptide encoded by IFN-L polypeptide allelic variants or IFN-Lpolypeptide splice variants, as defined herein.

The term “biologically active IFN-L polypeptides” refers to IFN-Lpolypeptides having at least one activity characteristic of thepolypeptide comprising the amino acid sequence of either SEQ ID NO: 2 orSEQ ID NO: 5. In addition, an IFN-L polypeptide may be active as animmunogen; that is, the IFN-L polypeptide contains at least one epitopeto which antibodies may be raised.

The term “isolated polypeptide” refers to a polypeptide of the presentinvention that (1) has been separated from at least about 50 percent ofpolynucleotides, lipids, carbohydrates, or other materials with which itis naturally found when isolated from the source cell, (2) is not linked(by covalent or noncovalent interaction) to all or a portion of apolypeptide to which the “isolated polypeptide” is linked in nature, (3)is operably linked (by covalent or noncovalent interaction) to apolypeptide with which it is not linked in nature, or (4) does not occurin nature. Preferably, the isolated polypeptide is substantially freefrom any other contaminating polypeptides or other contaminants that arefound in its natural environment that would interfere with itstherapeutic, diagnostic, prophylactic or research use.

The term “identity,” as known in the art, refers to a relationshipbetween the sequences of two or more polypeptide molecules or two ormore nucleic acid molecules, as determined by comparing the sequences.In the art, “identity” also means the degree of sequence relatednessbetween nucleic acid molecules or polypeptides, as the case may be, asdetermined by the match between strings of two or more nucleotide or twoor more amino acid sequences. “Identity” measures the percent ofidentical matches between the smaller of two or more sequences with gapalignments (if any) addressed by a particular mathematical model orcomputer program (i.e., “algorithms”).

The term “similarity” is a related concept, but in contrast to“identity,” “similarity” refers to a measure of relatedness whichincludes both identical matches and conservative substitution matches.If two polypeptide sequences have, for example, 10/20 identical aminoacids, and the remainder are all non-conservative substitutions, thenthe percent identity and similarity would both be 50%. If in the sameexample, there are five more positions where there are conservativesubstitutions, then the percent identity remains 50%, but the percentsimilarity would be 75% ( 15/20). Therefore, in cases where there areconservative substitutions, the percent similarity between twopolypeptides will be higher than the percent identity between those twopolypeptides.

The term “naturally occurring” or “native” when used in connection withbiological materials such as nucleic acid molecules, polypeptides, hostcells, and the like, refers to materials which are found in nature andare not manipulated by man. Similarly, “non-naturally occurring” or“non-native” as used herein refers to a material that is not found innature or that has been structurally modified or synthesized by man.

The terms “effective amount” and “therapeutically effective amount” eachrefer to the amount of an IFN-L polypeptide or IFN-L nucleic acidmolecule used to support an observable level of one or more biologicalactivities of the IFN-L polypeptides as set forth herein.

The term “pharmaceutically acceptable carrier” or “physiologicallyacceptable carrier” as used herein refers to one or more formulationmaterials suitable for accomplishing or enhancing the delivery of theIFN-L polypeptide, IFN-L nucleic acid molecule, or IFN-L selectivebinding agent as a pharmaceutical composition.

The term “antigen” refers to a molecule or a portion of a moleculecapable of being bound by a selective binding agent, such as anantibody, and additionally capable of being used in an animal to produceantibodies capable of binding to an epitope of that antigen. An antigenmay have one or more epitopes.

The term “selective binding agent” refers to a molecule or moleculeshaving specificity for an IFN-L polypeptide. As used herein, the terms,“specific” and “specificity” refer to the ability of the selectivebinding agents to bind to human IFN-L polypeptides and not to bind tohuman non-IFN-L polypeptides. It will be appreciated, however, that theselective binding agents may also bind orthologs of the polypeptide asset forth in either SEQ ID NO: 2 or SEQ ID NO: 5, that is, interspeciesversions thereof, such as mouse and rat IFN-L polypeptides.

The term “transduction” is used to refer to the transfer of genes fromone bacterium to another, usually by a phage. “Transduction” also refersto the acquisition and transfer of eukaryotic cellular sequences byretroviruses.

The term “transfection” is used to refer to the uptake of foreign orexogenous DNA by a cell, and a cell has been “transfected” when theexogenous DNA has been introduced inside the cell membrane. A number oftransfection techniques are well known in the art and are disclosedherein. See, e.g., Graham et al., 1973, Virology 52:456; Sambrook etal., Molecular Cloning, A Laboratory Manual (Cold Spring HarborLaboratories, 1989); Davis et al., Basic Methods in Molecular Biology(Elsevier, 1986); and Chu et al., 1981, Gene 13:197. Such techniques canbe used to introduce one or more exogenous DNA moieties into suitablehost cells.

The term “transformation” as used herein refers to a change in a cell'sgenetic characteristics, and a cell has been transformed when it hasbeen modified to contain a new DNA. For example, a cell is transformedwhere it is genetically modified from its native state. Followingtransfection or transduction, the transforming DNA may recombine withthat of the cell by physically integrating into a chromosome of thecell, may be maintained transiently as an episomal element without beingreplicated, or may replicate independently as a plasmid. A cell isconsidered to have been stably transformed when the DNA is replicatedwith the division of the cell.

Relatedness of Nucleic Acid Molecules and/or Polypeptides

It is understood that related nucleic acid molecules include allelic orsplice variants of the nucleic acid molecule of either SEQ ID NO: 1 orSEQ ID NO: 4, and include sequences which are complementary to any ofthe above nucleotide sequences. Related nucleic acid molecules alsoinclude a nucleotide sequence encoding a polypeptide comprising orconsisting essentially of a substitution, modification, addition and/ordeletion of one or more amino acid residues compared to the polypeptidein either SEQ ID NO: 2 or SEQ ID NO: 5. Such related IFN-L polypeptidesmay comprise, for example, an addition and/or a deletion of one or moreN-linked or O-linked glycosylation sites or an addition and/or adeletion of one or more cysteine residues.

Related nucleic acid molecules also include fragments of IFN-L nucleicacid molecules which encode a polypeptide of at least about 25contiguous amino acids, or about 50 amino acids, or about 75 aminoacids, or about 100 amino acids, or about 150 amino acids, or about 200amino acids, or more than 200 amino acid residues of the IFN-Lpolypeptide of either SEQ ID NO: 2 or SEQ ID NO: 5.

In addition, related IFN-L nucleic acid molecules also include thosemolecules which comprise nucleotide sequences which hybridize undermoderately or highly stringent conditions as defined herein with thefully complementary sequence of the IFN-L nucleic acid molecule ofeither SEQ ID NO: 1 or SEQ ID NO: 4, or of a molecule encoding apolypeptide, which polypeptide comprises the amino acid sequence asshown in either SEQ ID NO: 2 or SEQ ID NO: 5, or of a nucleic acidfragment as defined herein, or of a nucleic acid fragment encoding apolypeptide as defined herein. Hybridization probes may be preparedusing the IFN-L sequences provided herein to screen cDNA, genomic orsynthetic DNA libraries for related sequences. Regions of the DNA and/oramino acid sequence of IFN-L polypeptide that exhibit significantidentity to known sequences are readily determined using sequencealignment algorithms as described herein and those regions may be usedto design probes for screening.

The term “highly stringent conditions” refers to those conditions thatare designed to permit hybridization of DNA strands whose sequences arehighly complementary, and to exclude hybridization of significantlymismatched DNAs. Hybridization stringency is principally determined bytemperature, ionic strength, and the concentration of denaturing agentssuch as formamide. Examples of “highly stringent conditions” forhybridization and washing are 0.015 M sodium chloride, 0.0015 M sodiumcitrate at 65-68° C. or 0.015 M sodium chloride, 0.0015 M sodiumcitrate, and 50% formamide at 42° C. See Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring HarborLaboratory, 1989); Anderson et al., Nucleic Acid Hybridisation: APractical Approach Ch. 4 (IRL Press Limited).

More stringent conditions (such as higher temperature, lower ionicstrength, higher formamide, or other denaturing agent) may also beused—however, the rate of hybridization will be affected. Other agentsmay be included in the hybridization and washing buffers for the purposeof reducing non-specific and/or background hybridization. Examples are0.1% bovine serum albumin, 0.1% polyvinyl-pyrrolidone, 0.1% sodiumpyrophosphate, 0.1% sodium dodecylsulfate, NaDodSO₄, (SDS), ficoll,Denhardt's solution, sonicated salmon sperm DNA (or anothernon-complementary DNA), and dextran sulfate, although other suitableagents can also be used. The concentration and types of these additivescan be changed without substantially affecting the stringency of thehybridization conditions. Hybridization experiments are usually carriedout at pH 6.8-7.4; however, at typical ionic strength conditions, therate of hybridization is nearly independent of pH. See Anderson et al.,Nucleic Acid Hybridisation: A Practical Approach Ch. 4 (IRL PressLimited).

Factors affecting the stability of DNA duplex include base composition,length, and degree of base pair mismatch. Hybridization conditions canbe adjusted by one skilled in the art in order to accommodate thesevariables and allow DNAs of different sequence relatedness to formhybrids. The melting temperature of a perfectly matched DNA duplex canbe estimated by the following equation:

T _(m)(° C.)=81.5+16.6(log [Na ⁺])+0.41(% G+C)−600/N−0.72(% formamide)

where N is the length of the duplex formed, [Na+] is the molarconcentration of the sodium ion in the hybridization or washingsolution, % G+C is the percentage of (guanine+cytosine) bases in thehybrid. For imperfectly matched hybrids, the melting temperature isreduced by approximately 1° C. for each 1% mismatch.

The term “moderately stringent conditions” refers to conditions underwhich a DNA duplex with a greater degree of base pair mismatching thancould occur under “highly stringent conditions” is able to form.Examples of typical “moderately stringent conditions” are 0.015 M sodiumchloride, 0.0015 M sodium citrate at 50-65° C. or 0.015 M sodiumchloride, 0.0015 M sodium citrate, and 20% formamide at 37-50° C. By wayof example, “moderately stringent conditions” of 50° C. in 0.015 Msodium ion will allow about a 21% mismatch.

It will be appreciated by those skilled in the art that there is noabsolute distinction between “highly stringent conditions” and“moderately stringent conditions.” For example, at 0.015 M sodium ion(no formamide), the melting temperature of perfectly matched long DNA isabout 71° C. With a wash at 65° C. (at the same ionic strength), thiswould allow for approximately a 6% mismatch. To capture more distantlyrelated sequences, one skilled in the art can simply lower thetemperature or raise the ionic strength.

A good estimate of the melting temperature in 1M NaCl* foroligonucleotide probes up to about 20 nt is given by:

T _(m)=2° C. per A−T base pair+4° C. per G-C base pair

*The sodium ion concentration in 6× salt sodium citrate (SSC) is 1M. SeeSuggs et al., Developmental Biology Using Purified Genes 683 (Brown andFox, eds., 1981).

High stringency washing conditions for oligonucleotides are usually at atemperature of 0-5° C. below the Tm of the oligonucleotide in 6×SSC,0.1% SDS.

In another embodiment, related nucleic acid molecules comprise orconsist of a nucleotide sequence that is at least about 70 percentidentical to the nucleotide sequence as shown in either SEQ ID NO: 1 orSEQ ID NO: 4, or comprise or consist essentially of a nucleotidesequence encoding a polypeptide that is at least about 70 percentidentical to the polypeptide as set forth in either SEQ ID NO: 2 or SEQID NO: 5. In preferred embodiments, the nucleotide sequences are about75 percent, or about 80 percent, or about 85 percent, or about 90percent, or about 95, 96, 97, 98, or 99 percent identical to thenucleotide sequence as shown in either SEQ ID NO: 1 or SEQ ID NO: 4, orthe nucleotide sequences encode a polypeptide that is about 75 percent,or about 80 percent, or about 85 percent, or about 90 percent, or about95, 96, 97, 98, or 99 percent identical to the polypeptide sequence asset forth in either SEQ ID NO: 2 or SEQ ID NO: 5. Related nucleic acidmolecules encode polypeptides possessing at least one activity of thepolypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5.

Differences in the nucleic acid sequence may result in conservativeand/or non-conservative modifications of the amino acid sequencerelative to the amino acid sequence of either SEQ ID NO: 2 or SEQ ID NO:5.

Conservative modifications to the amino acid sequence of either SEQ IDNO: 2 or SEQ ID NO: 5 (and the corresponding modifications to theencoding nucleotides) will produce a polypeptide having functional andchemical characteristics similar to those of IFN-L polypeptides. Incontrast, substantial modifications in the functional and/or chemicalcharacteristics of IFN-L polypeptides may be accomplished by selectingsubstitutions in the amino acid sequence of either SEQ ID NO: 2 or SEQID NO: 5 that differ significantly in their effect on maintaining (a)the structure of the molecular backbone in the area of the substitution,for example, as a sheet or helical conformation, (b) the charge orhydrophobicity of the molecule at the target site, or (c) the bulk ofthe side chain.

For example, a “conservative amino acid substitution” may involve asubstitution of a native amino acid residue with a normative residuesuch that there is little or no effect on the polarity or charge of theamino acid residue at that position. Furthermore, any native residue inthe polypeptide may also be substituted with alanine, as has beenpreviously described for “alanine scanning mutagenesis.”

Conservative amino acid substitutions also encompass non-naturallyoccurring amino acid residues that are typically incorporated bychemical peptide synthesis rather than by synthesis in biologicalsystems. These include peptidomimetics, and other reversed or invertedforms of amino acid moieties.

Naturally occurring residues may be divided into classes based on commonside chain properties:

1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

2) neutral hydrophilic: Cys, Ser, Thr;

3) acidic: Asp, Glu;

4) basic: Asn, Gln, His, Lys, Arg;

5) residues that influence chain orientation: Gly, Pro; and

6) aromatic: Trp, Tyr, Phe.

For example, non-conservative substitutions may involve the exchange ofa member of one of these classes for a member from another class. Suchsubstituted residues may be introduced into regions of the human IFN-Lpolypeptide that are homologous with non-human IFN-L polypeptides, orinto the non-homologous regions of the molecule.

In making such changes, the hydropathic index of amino acids may beconsidered. Each amino acid has been assigned a hydropathic index on thebasis of its hydrophobicity and charge characteristics. The hydropathicindices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyte et al., 1982, J. Mol. Biol. 157:105-31). It is known thatcertain amino acids may be substituted for other amino acids having asimilar hydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, thesubstitution of amino acids whose hydropathic indices are within ±2 ispreferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functionally equivalent protein orpeptide thereby created is intended for use in immunologicalembodiments, as in the present case. The greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigenicity, i.e., with a biological property of the protein.

The following hydrophilicity values have been assigned to these aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1);glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5);histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine(−2.5); and tryptophan (−3.4). In making changes based upon similarhydrophilicity values, the substitution of amino acids whosehydrophilicity values are within ±2 is preferred, those which are within±1 are particularly preferred, and those within ±0.5 are even moreparticularly preferred. One may also identify epitopes from primaryamino acid sequences on the basis of hydrophilicity. These regions arealso referred to as “epitopic core regions.”

Desired amino acid substitutions (whether conservative ornon-conservative) can be determined by those skilled in the art at thetime such substitutions are desired. For example, amino acidsubstitutions can be used to identify important residues of the IFN-Lpolypeptide, or to increase or decrease the affinity of the IFN-Lpolypeptides described herein. Exemplary amino acid substitutions areset forth in Table I.

TABLE I Amino Acid Substitutions Original Residues ExemplarySubstitutions Preferred Substitutions Ala Val, Leu, Ile Val Arg Lys,Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn Asn GluAsp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met,Ala, Leu Phe, Norleucine Leu Norleucine, Ile, Ile Val, Met, Ala, Phe LysArg, 1,4 Diamino-butyric Arg Acid, Gln, Asn Met Leu, Phe, Ile Leu PheLeu, Val, Ile, Ala, Leu Tyr Pro Ala Gly Ser Thr, Ala, Cys Thr Thr SerSer Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe,Leu Ala, Norleucine

A skilled artisan will be able to determine suitable variants of thepolypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 usingwell-known techniques. For identifying suitable areas of the moleculethat may be changed without destroying biological activity, one skilledin the art may target areas not believed to be important for activity.For example, when similar polypeptides with similar activities from thesame species or from other species are known, one skilled in the art maycompare the amino acid sequence of an IFN-L polypeptide to such similarpolypeptides. With such a comparison, one can identify residues andportions of the molecules that are conserved among similar polypeptides.It will be appreciated that changes in areas of the IFN-L molecule thatare not conserved relative to such similar polypeptides would be lesslikely to adversely affect the biological activity and/or structure ofan IFN-L polypeptide. One skilled in the art would also know that, evenin relatively conserved regions, one may substitute chemically similaramino acids for the naturally occurring residues while retainingactivity (conservative amino acid residue substitutions). Therefore,even areas that may be important for biological activity or forstructure may be subject to conservative amino acid substitutionswithout destroying the biological activity or without adverselyaffecting the polypeptide structure.

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar polypeptides that are importantfor activity or structure. In view of such a comparison, one can predictthe importance of amino acid residues in an IFN-L polypeptide thatcorrespond to amino acid residues that are important for activity orstructure in similar polypeptides. One skilled in the art may opt forchemically similar amino acid substitutions for such predicted importantamino acid residues of IFN-L polypeptides.

One skilled in the art can also analyze the three-dimensional structureand amino acid sequence in relation to that structure in similarpolypeptides. In view of such information, one skilled in the art maypredict the alignment of amino acid residues of IFN-L polypeptide withrespect to its three dimensional structure. One skilled in the art maychoose not to make radical changes to amino acid residues predicted tobe on the surface of the protein, since such residues may be involved inimportant interactions with other molecules. Moreover, one skilled inthe art may generate test variants containing a single amino acidsubstitution at each amino acid residue. The variants could be screenedusing activity assays known to those with skill in the art. Suchvariants could be used to gather information about suitable variants.For example, if one discovered that a change to a particular amino acidresidue resulted in destroyed, undesirably reduced, or unsuitableactivity, variants with such a change would be avoided. In other words,based on information gathered from such routine experiments, one skilledin the art can readily determine the amino acids where furthersubstitutions should be avoided either alone or in combination withother mutations.

A number of scientific publications have been devoted to the predictionof secondary structure. See Moult, 1996, Curr. Opin. Biotechnol.7:422-27; Chou et al., 1974, Biochemistry 13:222-45; Chou et al., 1974,Biochemistry 113:211-22; Chou et al., 1978, Adv. Enzymol. Relat. AreasMol. Biol. 47:45-48; Chou et al., 1978, Ann. Rev. Biochem. 47:251-276;and Chou et al., 1979, Biophys. J. 26:367-84. Moreover, computerprograms are currently available to assist with predicting secondarystructure. One method of predicting secondary structure is based uponhomology modeling. For example, two polypeptides or proteins which havea sequence identity of greater than 30%, or similarity greater than 40%,often have similar structural topologies. The recent growth of theprotein structural database (PDB) has provided enhanced predictabilityof secondary structure, including the potential number of folds withinthe structure of a polypeptide or protein. See Holm et al., 1999,Nucleic Acids Res. 27:244-47. It has been suggested that there are alimited number of folds in a given polypeptide or protein and that oncea critical number of structures have been resolved, structuralprediction will become dramatically more accurate (Brenner et al., 1997,Curr. Opin. Struct. Biol. 7:369-76).

Additional methods of predicting secondary structure include “threading”(Jones, 1997, Curr. Opin. Struct. Biol. 7:377-87; Sippl et al., 1996,Structure 4:15-19), “profile analysis” (Bowie et al., 1991, Science,253:164-70; Gribskov et al., 1990, Methods Enzymol. 183:146-59; Gribskovet al., 1987, Proc. Nat. Acad. Sci. U.S.A. 84:4355-58), and“evolutionary linkage” (See Holm et al., supra, and Brenner et al.,supra).

Preferred IFN-L polypeptide variants include glycosylation variantswherein the number and/or type of glycosylation sites have been alteredcompared to the amino acid sequence set forth in either SEQ ID NO: 2 orSEQ ID NO: 5. In one embodiment, IFN-L polypeptide variants comprise agreater or a lesser number of N-linked glycosylation sites than theamino acid sequence set forth in either SEQ ID NO: 2 or SEQ ID NO: 5. AnN-linked glycosylation site is characterized by the sequence: Asn-X-Seror Asn-X-Thr, wherein the amino acid residue designated as X may be anyamino acid residue except proline. The substitution of amino acidresidues to create this sequence provides a potential new site for theaddition of an N-linked carbohydrate chain. Alternatively, substitutionsthat eliminate this sequence will remove an existing N-linkedcarbohydrate chain. Also provided is a rearrangement of N-linkedcarbohydrate chains wherein one or more N-linked glycosylation sites(typically those that are naturally occurring) are eliminated and one ormore new N-linked sites are created. Additional preferred IFN-L variantsinclude cysteine variants, wherein one or more cysteine residues aredeleted or substituted with another amino acid (e.g., serine) ascompared to the amino acid sequence set forth in either SEQ ID NO: 2 orSEQ ID NO: 5. Cysteine variants are useful when IFN-L polypeptides mustbe refolded into a biologically active conformation such as after theisolation of insoluble inclusion bodies. Cysteine variants generallyhave fewer cysteine residues than the native protein, and typically havean even number to minimize interactions resulting from unpairedcysteines.

In other embodiments, related nucleic acid molecules comprise or consistof a nucleotide sequence encoding a polypeptide as set forth in eitherSEQ ID NO: 2 or SEQ ID NO: 5 with at least one amino acid insertion andwherein the polypeptide has an activity of the polypeptide set forth ineither SEQ ID NO: 2 or SEQ ID NO: 5, or a nucleotide sequence encoding apolypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 with atleast one amino acid deletion and wherein the polypeptide has anactivity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ IDNO: 5. Related nucleic acid molecules also comprise or consist of anucleotide sequence encoding a polypeptide as set forth in either SEQ IDNO: 2 or SEQ ID NO: 5 wherein the polypeptide has a carboxyl- and/oramino-terminal truncation and further wherein the polypeptide has anactivity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ IDNO: 5. Related nucleic acid molecules also comprise or consist of anucleotide sequence encoding a polypeptide as set forth in either SEQ IDNO: 2 or SEQ ID NO: 5 with at least one modification selected from thegroup consisting of amino acid substitutions, amino acid insertions,amino acid deletions, carboxyl-terminal truncations, and amino-terminaltruncations and wherein the polypeptide has an activity of thepolypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5.

In addition, the polypeptide comprising the amino acid sequence ofeither SEQ ID NO: 2 or SEQ ID NO: 5, or other IFN-L polypeptide, may befused to a homologous polypeptide to form a homodimer or to aheterologous polypeptide to form a heterodimer Heterologous peptides andpolypeptides include, but are not limited to: an epitope to allow forthe detection and/or isolation of an IFN-L fusion polypeptide; atransmembrane receptor protein or a portion thereof, such as anextracellular domain or a transmembrane and intracellular domain; aligand or a portion thereof which binds to a transmembrane receptorprotein; an enzyme or portion thereof which is catalytically active; apolypeptide or peptide which promotes oligomerization, such as a leucinezipper domain; a polypeptide or peptide which increases stability, suchas an immunoglobulin constant region; and a polypeptide which has atherapeutic activity different from the polypeptide comprising the aminoacid sequence as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5, orother IFN-L polypeptide.

Fusions can be made either at the amino-terminus or at thecarboxyl-terminus of the polypeptide comprising the amino acid sequenceset forth in either SEQ ID NO: 2 or SEQ ID NO: 5, or other IFN-Lpolypeptide. Fusions may be direct with no linker or adapter molecule ormay be through a linker or adapter molecule. A linker or adaptermolecule may be one or more amino acid residues, typically from about 20to about 50 amino acid residues. A linker or adapter molecule may alsobe designed with a cleavage site for a DNA restriction endonuclease orfor a protease to allow for the separation of the fused moieties. Itwill be appreciated that once constructed, the fusion polypeptides canbe derivatized according to the methods described herein.

In a further embodiment of the invention, the polypeptide comprising theamino acid sequence of either SEQ ID NO: 2 or SEQ ID NO: 5, or otherIFN-L polypeptide, is fused to one or more domains of an Fc region ofhuman IgG. Antibodies comprise two functionally independent parts, avariable domain known as “Fab,” that binds an antigen, and a constantdomain known as “Fc,” that is involved in effector functions such ascomplement activation and attack by phagocytic cells. An Fc has a longserum half-life, whereas an Fab is short-lived. Capon et al., 1989,Nature 337:525-31. When constructed together with a therapeutic protein,an Fc domain can provide longer half-life or incorporate such functionsas Fc receptor binding, protein A binding, complement fixation, andperhaps even placental transfer. Id. Table II summarizes the use ofcertain Fc fusions known in the art.

TABLE II Fc Fusion with Therapeutic Proteins Form of Fc Fusion partnerTherapeutic implications Reference IgG1 N-terminus of Hodgkin's disease;U.S. Pat. No. CD30-L anaplastic lymphoma; T- 5,480,981 cell leukemiaMurine Fcγ2a IL-10 anti-inflammatory; Zheng et al., 1995, J. transplantrejection Immunol. 154: 5590-600 IgG1 TNF receptor septic shock Fisheret al., 1996, N. Engl. J. Med. 334: 1697-1702; Van Zee et al., 1996, J.Immunol. 156: 2221-30 IgG, IgA, IgM, TNF receptor inflammation, U.S.Pat. No. or IgE autoimmune disorders 5,808,029 (excluding the firstdomain) IgG1 CD4 receptor AIDS Capon et al., 1989, Nature 337: 525-31IgG1, N-terminus anti-cancer, antiviral Harvill et al., 1995, IgG3 ofIL-2 Immunotech. 1: 95-105 IgG1 C-terminus of osteoarthritis; WO97/23614 OPG bone density IgG1 N-terminus of anti-obesity PCT/US97/23183, filed leptin Dec. 11, 1997 Human Ig Cγ1 CTLA-4 autoimmunedisorders Linsley, 1991, J. Exp. Med., 174: 561-69

In one example, a human IgG hinge, CH2, and CH3 region may be fused ateither the amino-terminus or carboxyl-terminus of the IFN-L polypeptidesusing methods known to the skilled artisan. In another example, a humanIgG hinge, CH2, and CH3 region may be fused at either the amino-terminusor carboxyl-terminus of an IFN-L polypeptide fragment (e.g., thepredicted extracellular portion of IFN-L polypeptide).

The resulting IFN-L fusion polypeptide may be purified by use of aProtein A affinity column. Peptides and proteins fused to an Fc regionhave been found to exhibit a substantially greater half-life in vivothan the unfused counterpart. Also, a fusion to an Fc region allows fordimerization/multimerization of the fusion polypeptide. The Fc regionmay be a naturally occurring Fc region, or may be altered to improvecertain qualities, such as therapeutic qualities, circulation time, orreduced aggregation.

Identity and similarity of related nucleic acid molecules andpolypeptides are readily calculated by known methods. Such methodsinclude, but are not limited to those described in ComputationalMolecular Biology (A. M. Lesk, ed., Oxford University Press 1988);Biocomputing: Informatics and Genome Projects (D. W. Smith, ed.,Academic Press 1993); Computer Analysis of Sequence Data (Part 1, A. M.Griffin and H. G. Griffin, eds., Humana Press 1994); G. von Heinle,Sequence Analysis in Molecular Biology (Academic Press 1987); SequenceAnalysis Primer (M. Gribskov and J. Devereux, eds., M. Stockton Press1991); and Carillo et al., 1988, SIAM J. Applied Math., 48:1073.

Preferred methods to determine identity and/or similarity are designedto give the largest match between the sequences tested. Methods todetermine identity and similarity are described in publicly availablecomputer programs. Preferred computer program methods to determineidentity and similarity between two sequences include, but are notlimited to, the GCG program package, including GAP (Devereux et al.,1984, Nucleic Acids Res. 12:387; Genetics Computer Group, University ofWisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al.,1990, J. Mol. Biol. 215:403-10). The BLASTX program is publiclyavailable from the National Center for Biotechnology Information (NCBI)and other sources (Altschul et al., BLAST Manual (NCB NLM NIH, Bethesda,Md.); Altschul et al., 1990, supra). The well-known Smith Watermanalgorithm may also be used to determine identity.

Certain alignment schemes for aligning two amino acid sequences mayresult in the matching of only a short region of the two sequences, andthis small aligned region may have very high sequence identity eventhough there is no significant relationship between the two full-lengthsequences. Accordingly, in a preferred embodiment, the selectedalignment method (GAP program) will result in an alignment that spans atleast 50 contiguous amino acids of the claimed polypeptide.

For example, using the computer algorithm GAP (Genetics Computer Group,University of Wisconsin, Madison, Wis.), two polypeptides for which thepercent sequence identity is to be determined are aligned for optimalmatching of their respective amino acids (the “matched span,” asdetermined by the algorithm). A gap opening penalty (which is calculatedas 3× the average diagonal; the “average diagonal” is the average of thediagonal of the comparison matrix being used; the “diagonal” is thescore or number assigned to each perfect amino acid match by theparticular comparison matrix) and a gap extension penalty (which isusually 0.1× the gap opening penalty), as well as a comparison matrixsuch as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm.A standard comparison matrix is also used by the algorithm (see Dayhoffet al., 5 Atlas of Protein Sequence and Structure (Supp. 3 1978)(PAM250comparison matrix); Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA89:10915-19 (BLOSUM 62 comparison matrix)).

Preferred parameters for polypeptide sequence comparison include thefollowing:

Algorithm: Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-53;

Comparison matrix: BLOSUM 62 (Henikoff et al., supra);

Gap Penalty: 12

Gap Length Penalty: 4

Threshold of Similarity: 0

The GAP program is useful with the above parameters. The aforementionedparameters are the default parameters for polypeptide comparisons (alongwith no penalty for end gaps) using the GAP algorithm.

Preferred parameters for nucleic acid molecule sequence comparisoninclude the following:

Algorithm: Needleman and Wunsch, supra;

Comparison matrix: matches=+10, mismatch=0

Gap Penalty: 50

Gap Length Penalty: 3

The GAP program is also useful with the above parameters. Theaforementioned parameters are the default parameters for nucleic acidmolecule comparisons.

Other exemplary algorithms, gap opening penalties, gap extensionpenalties, comparison matrices, and thresholds of similarity may beused, including those set forth in the Program Manual, WisconsinPackage, Version 9, September, 1997. The particular choices to be madewill be apparent to those of skill in the art and will depend on thespecific comparison to be made, such as DNA-to-DNA, protein-to-protein,protein-to-DNA; and additionally, whether the comparison is betweengiven pairs of sequences (in which case GAP or BestFit are generallypreferred) or between one sequence and a large database of sequences (inwhich case FASTA or BLASTA are preferred).

Nucleic Acid Molecules

The nucleic acid molecules encoding a polypeptide comprising the aminoacid sequence of an IFN-L polypeptide can readily be obtained in avariety of ways including, without limitation, chemical synthesis, cDNAor genomic library screening, expression library screening, and/or PCRamplification of cDNA.

Recombinant DNA methods used herein are generally those set forth inSambrook et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory Press, 1989) and/or Current Protocols in MolecularBiology (Ausubel et al., eds., Green Publishers Inc. and Wiley and Sons1994). The invention provides for nucleic acid molecules as describedherein and methods for obtaining such molecules.

Where a gene encoding the amino acid sequence of an IFN-L polypeptidehas been identified from one species, all or a portion of that gene maybe used as a probe to identify orthologs or related genes from the samespecies. The probes or primers may be used to screen cDNA libraries fromvarious tissue sources believed to express the IFN-L polypeptide. Inaddition, part or all of a nucleic acid molecule having the sequence asset forth in either SEQ ID NO: 1 or SEQ ID NO: 4 may be used to screen agenomic library to identify and isolate a gene encoding the amino acidsequence of an IFN-L polypeptide. Typically, conditions of moderate orhigh stringency will be employed for screening to minimize the number offalse positives obtained from the screening.

Nucleic acid molecules encoding the amino acid sequence of IFN-Lpolypeptides may also be identified by expression cloning which employsthe detection of positive clones based upon a property of the expressedprotein. Typically, nucleic acid libraries are screened by the bindingan antibody or other binding partner (e.g., receptor or ligand) tocloned proteins that are expressed and displayed on a host cell surface.The antibody or binding partner is modified with a detectable label toidentify those cells expressing the desired clone.

Recombinant expression techniques conducted in accordance with thedescriptions set forth below may be followed to produce thesepolynucleotides and to express the encoded polypeptides. For example, byinserting a nucleic acid sequence that encodes the amino acid sequenceof an IFN-L polypeptide into an appropriate vector, one skilled in theart can readily produce large quantities of the desired nucleotidesequence. The sequences can then be used to generate detection probes oramplification primers. Alternatively, a polynucleotide encoding theamino acid sequence of an IFN-L polypeptide can be inserted into anexpression vector. By introducing the expression vector into anappropriate host, the encoded IFN-L polypeptide may be produced in largeamounts.

Another method for obtaining a suitable nucleic acid sequence is thepolymerase chain reaction (PCR). In this method, cDNA is prepared frompoly(A)+RNA or total RNA using the enzyme reverse transcriptase. Twoprimers, typically complementary to two separate regions of cDNAencoding the amino acid sequence of an IFN-L polypeptide, are then addedto the cDNA along with a polymerase such as Taq polymerase, and thepolymerase amplifies the cDNA region between the two primers.

Another means of preparing a nucleic acid molecule encoding the aminoacid sequence of an IFN-L polypeptide is chemical synthesis usingmethods well known to the skilled artisan such as those described byEngels et al., 1989, Angew. Chem. Intl. Ed. 28:716-34. These methodsinclude, inter alia, the phosphotriester, phosphoramidite, andH-phosphonate methods for nucleic acid synthesis. A preferred method forsuch chemical synthesis is polymer-supported synthesis using standardphosphoramidite chemistry. Typically, the DNA encoding the amino acidsequence of an IFN-L polypeptide will be several hundred nucleotides inlength. Nucleic acids larger than about 100 nucleotides can besynthesized as several fragments using these methods. The fragments canthen be ligated together to form the full-length nucleotide sequence ofan IFN-L gene. Usually, the DNA fragment encoding the amino-terminus ofthe polypeptide will have an ATG, which encodes a methionine residue.This methionine may or may not be present on the mature form of theIFN-L polypeptide, depending on whether the polypeptide produced in thehost cell is designed to be secreted from that cell. Other methods knownto the skilled artisan may be used as well.

In certain embodiments, nucleic acid variants contain codons which havebeen altered for optimal expression of an IFN-L polypeptide in a givenhost cell. Particular codon alterations will depend upon the IFN-Lpolypeptide and host cell selected for expression. Such “codonoptimization” can be carried out by a variety of methods, for example,by selecting codons which are preferred for use in highly expressedgenes in a given host cell. Computer algorithms which incorporate codonfrequency tables such as “Eco_high.Cod” for codon preference of highlyexpressed bacterial genes may be used and are provided by the Universityof Wisconsin Package Version 9.0 (Genetics Computer Group, Madison,Wis.). Other useful codon frequency tables include “Celeganshigh.cod,”“Celegans_low.cod,” “Drosophila_high.cod,” “Human_high.cod,”“Maize_high.cod,” and “Yeast_high.cod.”

In some cases, it may be desirable to prepare nucleic acid moleculesencoding IFN-L polypeptide variants. Nucleic acid molecules encodingvariants may be produced using site directed mutagenesis, PCRamplification, or other appropriate methods, where the primer(s) havethe desired point mutations (see Sambrook et al., supra, and Ausubel etal., supra, for descriptions of mutagenesis techniques). Chemicalsynthesis using methods described by Engels et al., supra, may also beused to prepare such variants. Other methods known to the skilledartisan may be used as well.

Vectors and Host Cells

A nucleic acid molecule encoding the amino acid sequence of an IFN-Lpolypeptide is inserted into an appropriate expression vector usingstandard ligation techniques. The vector is typically selected to befunctional in the particular host cell employed (i.e., the vector iscompatible with the host cell machinery such that amplification of thegene and/or expression of the gene can occur). A nucleic acid moleculeencoding the amino acid sequence of an IFN-L polypeptide may beamplified/expressed in prokaryotic, yeast, insect (baculovirus systems)and/or eukaryotic host cells. Selection of the host cell will depend inpart on whether an IFN-L polypeptide is to be post-translationallymodified (e.g., glycosylated and/or phosphorylated). If so, yeast,insect, or mammalian host cells are preferable. For a review ofexpression vectors, see Meth. Enz., vol. 185 (D. V. Goeddel, ed.,Academic Press 1990).

Typically, expression vectors used in any of the host cells will containsequences for plasmid maintenance and for cloning and expression ofexogenous nucleotide sequences. Such sequences, collectively referred toas “flanking sequences” in certain embodiments will typically includeone or more of the following nucleotide sequences: a promoter, one ormore enhancer sequences, an origin of replication, a transcriptionaltermination sequence, a complete intron sequence containing a donor andacceptor splice site, a sequence encoding a leader sequence forpolypeptide secretion, a ribosome binding site, a polyadenylationsequence, a polylinker region for inserting the nucleic acid encodingthe polypeptide to be expressed, and a selectable marker element. Eachof these sequences is discussed below.

Optionally, the vector may contain a “tag”-encoding sequence, i.e., anoligonucleotide molecule located at the 5′ or 3′ end of the IFN-Lpolypeptide coding sequence; the oligonucleotide sequence encodespolyHis (such as hexaHis), or another “tag” such as FLAG, HA(hemaglutinin influenza virus), or myc for which commercially availableantibodies exist. This tag is typically fused to the polypeptide uponexpression of the polypeptide, and can serve as a means for affinitypurification of the IFN-L polypeptide from the host cell. Affinitypurification can be accomplished, for example, by column chromatographyusing antibodies against the tag as an affinity matrix. Optionally, thetag can subsequently be removed from the purified IFN-L polypeptide byvarious means such as using certain peptidases for cleavage.

Flanking sequences may be homologous (i.e., from the same species and/orstrain as the host cell), heterologous (i.e., from a species other thanthe host cell species or strain), hybrid (i.e., a combination offlanking sequences from more than one source), or synthetic, or theflanking sequences may be native sequences which normally function toregulate IFN-L polypeptide expression. As such, the source of a flankingsequence may be any prokaryotic or eukaryotic organism, any vertebrateor invertebrate organism, or any plant, provided that the flankingsequence is functional in, and can be activated by, the host cellmachinery.

Flanking sequences useful in the vectors of this invention may beobtained by any of several methods well known in the art. Typically,flanking sequences useful herein—other than the IFN-L gene flankingsequences—will have been previously identified by mapping and/or byrestriction endonuclease digestion and can thus be isolated from theproper tissue source using the appropriate restriction endonucleases. Insome cases, the full nucleotide sequence of a flanking sequence may beknown. Here, the flanking sequence may be synthesized using the methodsdescribed herein for nucleic acid synthesis or cloning.

Where all or only a portion of the flanking sequence is known, it may beobtained using PCR and/or by screening a genomic library with a suitableoligonucleotide and/or flanking sequence fragment from the same oranother species. Where the flanking sequence is not known, a fragment ofDNA containing a flanking sequence may be isolated from a larger pieceof DNA that may contain, for example, a coding sequence or even anothergene or genes. Isolation may be accomplished by restriction endonucleasedigestion to produce the proper DNA fragment followed by isolation usingagarose gel purification, Qiagen® column chromatography (Chatsworth,Calif.), or other methods known to the skilled artisan. The selection ofsuitable enzymes to accomplish this purpose will be readily apparent toone of ordinary skill in the art.

An origin of replication is typically a part of those prokaryoticexpression vectors purchased commercially, and the origin aids in theamplification of the vector in a host cell. Amplification of the vectorto a certain copy number can, in some cases, be important for theoptimal expression of an IFN-L polypeptide. If the vector of choice doesnot contain an origin of replication site, one may be chemicallysynthesized based on a known sequence, and ligated into the vector. Forexample, the origin of replication from the plasmid pBR322 (New EnglandBiolabs, Beverly, Mass.) is suitable for most gram-negative bacteria andvarious origins (e.g., SV40, polyoma, adenovirus, vesicular stomatitusvirus (VSV), or papillomaviruses such as HPV or BPV) are useful forcloning vectors in mammalian cells. Generally, the origin of replicationcomponent is not needed for mammalian expression vectors (for example,the SV40 origin is often used only because it contains the earlypromoter).

A transcription termination sequence is typically located 3′ of the endof a polypeptide coding region and serves to terminate transcription.Usually, a transcription termination sequence in prokaryotic cells is aG-C rich fragment followed by a poly-T sequence. While the sequence iseasily cloned from a library or even purchased commercially as part of avector, it can also be readily synthesized using methods for nucleicacid synthesis such as those described herein.

A selectable marker gene element encodes a protein necessary for thesurvival and growth of a host cell grown in a selective culture medium.Typical selection marker genes encode proteins that (a) conferresistance to antibiotics or other toxins, e.g., ampicillin,tetracycline, or kanamycin for prokaryotic host cells; (b) complementauxotrophic deficiencies of the cell; or (c) supply critical nutrientsnot available from complex media. Preferred selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene. A neomycin resistance gene may also beused for selection in prokaryotic and eukaryotic host cells.

Other selection genes may be used to amplify the gene that will beexpressed. Amplification is the process wherein genes that are ingreater demand for the production of a protein critical for growth arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Examples of suitable selectable markers for mammaliancells include dihydrofolate reductase (DHFR) and thymidine kinase. Themammalian cell transformants are placed under selection pressure whereinonly the transformants are uniquely adapted to survive by virtue of theselection gene present in the vector. Selection pressure is imposed byculturing the transformed cells under conditions in which theconcentration of selection agent in the medium is successively changed,thereby leading to the amplification of both the selection gene and theDNA that encodes an IFN-L polypeptide. As a result, increased quantitiesof IFN-L polypeptide are synthesized from the amplified DNA.

A ribosome binding site is usually necessary for translation initiationof mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes)or a Kozak sequence (eukaryotes). The element is typically located 3′ tothe promoter and 5′ to the coding sequence of an IFN-L polypeptide to beexpressed. The Shine-Dalgarno sequence is varied but is typically apolypurine (i.e., having a high A-G content). Many Shine-Dalgarnosequences have been identified, each of which can be readily synthesizedusing methods set forth herein and used in a prokaryotic vector.

A leader, or signal, sequence may be used to direct an IFN-L polypeptideout of the host cell. Typically, a nucleotide sequence encoding thesignal sequence is positioned in the coding region of an IFN-L nucleicacid molecule, or directly at the 5′ end of an IFN-L polypeptide codingregion. Many signal sequences have been identified, and any of thosethat are functional in the selected host cell may be used in conjunctionwith an IFN-L nucleic acid molecule. Therefore, a signal sequence may behomologous (naturally occurring) or heterologous to the IFN-L nucleicacid molecule. Additionally, a signal sequence may be chemicallysynthesized using methods described herein. In most cases, the secretionof an IFN-L polypeptide from the host cell via the presence of a signalpeptide will result in the removal of the signal peptide from thesecreted IFN-L polypeptide. The signal sequence may be a component ofthe vector, or it may be a part of an IFN-L nucleic acid molecule thatis inserted into the vector.

Included within the scope of this invention is the use of either anucleotide sequence encoding a native IFN-L polypeptide signal sequencejoined to an IFN-L polypeptide coding region or a nucleotide sequenceencoding a heterologous signal sequence joined to an IFN-L polypeptidecoding region. The heterologous signal sequence selected should be onethat is recognized and processed, i.e., cleaved by a signal peptidase,by the host cell. For prokaryotic host cells that do not recognize andprocess the native IFN-L polypeptide signal sequence, the signalsequence is substituted by a prokaryotic signal sequence selected, forexample, from the group of the alkaline phosphatase, penicillinase, orheat-stable enterotoxin II leaders. For yeast secretion, the nativeIFN-L polypeptide signal sequence may be substituted by the yeastinvertase, alpha factor, or acid phosphatase leaders. In mammalian cellexpression the native signal sequence is satisfactory, although othermammalian signal sequences may be suitable.

In some cases, such as where glycosylation is desired in a eukaryotichost cell expression system, one may manipulate the various presequencesto improve glycosylation or yield. For example, one may alter thepeptidase cleavage site of a particular signal peptide, or addpro-sequences, which also may affect glycosylation. The final proteinproduct may have, in the −1 position (relative to the first amino acidof the mature protein) one or more additional amino acids incident toexpression, which may not have been totally removed. For example, thefinal protein product may have one or two amino acid residues found inthe peptidase cleavage site, attached to the amino-terminus.Alternatively, use of some enzyme cleavage sites may result in aslightly truncated form of the desired IFN-L polypeptide, if the enzymecuts at such area within the mature polypeptide.

In many cases, transcription of a nucleic acid molecule is increased bythe presence of one or more introns in the vector; this is particularlytrue where a polypeptide is produced in eukaryotic host cells,especially mammalian host cells. The introns used may be naturallyoccurring within the IFN-L gene especially where the gene used is afull-length genomic sequence or a fragment thereof. Where the intron isnot naturally occurring within the gene (as for most cDNAs), the intronmay be obtained from another source. The position of the intron withrespect to flanking sequences and the IFN-L gene is generally important,as the intron must be transcribed to be effective. Thus, when an IFN-LcDNA molecule is being transcribed, the preferred position for theintron is 3′ to the transcription start site and 5′ to the poly-Atranscription termination sequence. Preferably, the intron or intronswill be located on one side or the other (i.e., 5′ or 3′) of the cDNAsuch that it does not interrupt the coding sequence. Any intron from anysource, including viral, prokaryotic and eukaryotic (plant or animal)organisms, may be used to practice this invention, provided that it iscompatible with the host cell into which it is inserted. Also includedherein are synthetic introns. Optionally, more than one intron may beused in the vector.

The expression and cloning vectors of the present invention willtypically contain a promoter that is recognized by the host organism andoperably linked to the molecule encoding the IFN-L polypeptide.Promoters are untranscribed sequences located upstream (i.e., 5′) to thestart codon of a structural gene (generally within about 100 to 1000 bp)that control the transcription of the structural gene. Promoters areconventionally grouped into one of two classes: inducible promoters andconstitutive promoters. Inducible promoters initiate increased levels oftranscription from DNA under their control in response to some change inculture conditions, such as the presence or absence of a nutrient or achange in temperature. Constitutive promoters, on the other hand,initiate continual gene product production; that is, there is little orno control over gene expression. A large number of promoters, recognizedby a variety of potential host cells, are well known. A suitablepromoter is operably linked to the DNA encoding IFN-L polypeptide byremoving the promoter from the source DNA by restriction enzymedigestion and inserting the desired promoter sequence into the vector.The native IFN-L promoter sequence may be used to direct amplificationand/or expression of an IFN-L nucleic acid molecule. A heterologouspromoter is preferred, however, if it permits greater transcription andhigher yields of the expressed protein as compared to the nativepromoter, and if it is compatible with the host cell system that hasbeen selected for use.

Promoters suitable for use with prokaryotic hosts include thebeta-lactamase and lactose promoter systems; alkaline phosphatase; atryptophan (trp) promoter system; and hybrid promoters such as the tacpromoter. Other known bacterial promoters are also suitable. Theirsequences have been published, thereby enabling one skilled in the artto ligate them to the desired DNA sequence, using linkers or adapters asneeded to supply any useful restriction sites.

Suitable promoters for use with yeast hosts are also well known in theart. Yeast enhancers are advantageously used with yeast promoters.Suitable promoters for use with mammalian host cells are well known andinclude, but are not limited to, those obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, retroviruses, hepatitis-B virus and most preferablySimian Virus 40 (SV40). Other suitable mammalian promoters includeheterologous mammalian promoters, for example, heat-shock promoters andthe actin promoter.

Additional promoters which may be of interest in controlling IFN-L geneexpression include, but are not limited to: the SV40 early promoterregion (Bernoist and Chambon, 1981, Nature 290:304-10); the CMVpromoter; the promoter contained in the 3′ long terminal repeat of Roussarcoma virus (Yamamoto, et al., 1980, Cell 22:787-97); the herpesthymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci.U.S.A. 78:1444-45); the regulatory sequences of the metallothionine gene(Brinster et al., 1982, Nature 296:39-42); prokaryotic expressionvectors such as the beta-lactamase promoter (Villa-Kamaroff et al.,1978, Proc. Natl. Acad. Sci. U.S.A., 75:3727-31); or the tac promoter(DeBoer et al., 1983, Proc. Natl. Acad. Sci. U.S.A., 80:21-25). Also ofinterest are the following animal transcriptional control regions, whichexhibit tissue specificity and have been utilized in transgenic animals:the elastase I gene control region which is active in pancreatic acinarcells (Swift et al., 1984, Cell 38:639-46; Ornitz et al., 1986, ColdSpring Harbor Symp. Quant. Biol. 50:399-409 (1986); MacDonald, 1987,Hepatology 7:425-515); the insulin gene control region which is activein pancreatic beta cells (Hanahan, 1985, Nature 315:115-22); theimmunoglobulin gene control region which is active in lymphoid cells(Grosschedl et al., 1984, Cell 38:647-58; Adames et al., 1985, Nature318:533-38; Alexander et al., 1987, Mol. Cell. Biol., 7:1436-44); themouse mammary tumor virus control region which is active in testicular,breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-95);the albumin gene control region which is active in liver (Pinkert etal., 1987, Genes and Devel. 1:268-76); the alpha-feto-protein genecontrol region which is active in liver (Krumlauf et al., 1985, Mol.Cell. Biol., 5:1639-48; Hammer et al., 1987, Science 235:53-58); thealpha 1-antitrypsin gene control region which is active in the liver(Kelsey et al., 1987, Genes and Devel. 1:161-71); the beta-globin genecontrol region which is active in myeloid cells (Mogram et al., 1985,Nature 315:338-40; Kollias et al., 1986, Cell 46:89-94); the myelinbasic protein gene control region which is active in oligodendrocytecells in the brain (Readhead et al., 1987, Cell 48:703-12); the myosinlight chain-2 gene control region which is active in skeletal muscle(Sani, 1985, Nature 314:283-86); and the gonadotropic releasing hormonegene control region which is active in the hypothalamus (Mason et al.,1986, Science 234:1372-78).

An enhancer sequence may be inserted into the vector to increase thetranscription of a DNA encoding an IFN-L polypeptide of the presentinvention by higher eukaryotes. Enhancers are cis-acting elements ofDNA, usually about 10-300 bp in length, that act on the promoter toincrease transcription. Enhancers are relatively orientation andposition independent. They have been found 5′ and 3′ to thetranscription unit. Several enhancer sequences available from mammaliangenes are known (e.g., globin, elastase, albumin, alpha-feto-protein andinsulin). Typically, however, an enhancer from a virus will be used. TheSV40 enhancer, the cytomegalovirus early promoter enhancer, the polyomaenhancer, and adenovirus enhancers are exemplary enhancing elements forthe activation of eukaryotic promoters. While an enhancer may be splicedinto the vector at a position 5′ or 3′ to an IFN-L nucleic acidmolecule, it is typically located at a site 5′ from the promoter.

Expression vectors of the invention may be constructed from a startingvector such as a commercially available vector. Such vectors may or maynot contain all of the desired flanking sequences. Where one or more ofthe flanking sequences described herein are not already present in thevector, they may be individually obtained and ligated into the vector.Methods used for obtaining each of the flanking sequences are well knownto one skilled in the art.

Preferred vectors for practicing this invention are those which arecompatible with bacterial, insect, and mammalian host cells. Suchvectors include, inter alia, pCRII, pCR3, and pcDNA3.1 (Invitrogen, SanDiego, Calif.), pBSII (Stratagene, La Jolla, Calif.), pET15 (Novagen,Madison, Wis.), pGEX (Pharmacia Biotech, Piscataway, N.J.), pEGFP-N2(Clontech, Palo Alto, Calif.), pETL (BlueBacII, Invitrogen), pDSR-alpha(PCT Pub. No. WO 90/14363) and pFastBacDual (Gibco-BRL, Grand Island,N.Y.).

Additional suitable vectors include, but are not limited to, cosmids,plasmids, or modified viruses, but it will be appreciated that thevector system must be compatible with the selected host cell. Suchvectors include, but are not limited to plasmids such as Bluescript®plasmid derivatives (a high copy number ColE1-based phagemid, StratageneCloning Systems, La Jolla Calif.), PCR cloning plasmids designed forcloning Taq-amplified PCR products (e.g., TOPO™ TA Cloning® Kit, PCR2.1®plasmid derivatives, Invitrogen, Carlsbad, Calif.), and mammalian, yeastor virus vectors such as a baculovirus expression system (pBacPAKplasmid derivatives, Clontech, Palo Alto, Calif.).

After the vector has been constructed and a nucleic acid moleculeencoding an IFN-L polypeptide has been inserted into the proper site ofthe vector, the completed vector may be inserted into a suitable hostcell for amplification and/or polypeptide expression. The transformationof an expression vector for an IFN-L polypeptide into a selected hostcell may be accomplished by well known methods including methods such astransfection, infection, calcium chloride, electroporation,microinjection, lipofection, DEAE-dextran method, or other knowntechniques. The method selected will in part be a function of the typeof host cell to be used. These methods and other suitable methods arewell known to the skilled artisan, and are set forth, for example, inSambrook et al., supra.

Host cells may be prokaryotic host cells (such as E. coli) or eukaryotichost cells (such as a yeast, insect, or vertebrate cell). The host cell,when cultured under appropriate conditions, synthesizes an IFN-Lpolypeptide which can subsequently be collected from the culture medium(if the host cell secretes it into the medium) or directly from the hostcell producing it (if it is not secreted). The selection of anappropriate host cell will depend upon various factors, such as desiredexpression levels, polypeptide modifications that are desirable ornecessary for activity (such as glycosylation or phosphorylation) andease of folding into a biologically active molecule.

A number of suitable host cells are known in the art and many areavailable from the American Type Culture Collection (ATCC), Manassas,Va. Examples include, but are not limited to, mammalian cells, such asChinese hamster ovary cells (CHO), CHO DHFR(−) cells (Urlaub et al.,1980, Proc. Natl. Acad. Sci. U.S.A. 97:4216-20), human embryonic kidney(HEK) 293 or 293T cells, or 3T3 cells. The selection of suitablemammalian host cells and methods for transformation, culture,amplification, screening, product production, and purification are knownin the art. Other suitable mammalian cell lines, are the monkey COS-1and COS-7 cell lines, and the CV-1 cell line. Further exemplarymammalian host cells include primate cell lines and rodent cell lines,including transformed cell lines. Normal diploid cells, cell strainsderived from in vitro culture of primary tissue, as well as primaryexplants, are also suitable. Candidate cells may be genotypicallydeficient in the selection gene, or may contain a dominantly actingselection gene. Other suitable mammalian cell lines include but are notlimited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929 cells, 3T3lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster celllines. Each of these cell lines is known by and available to thoseskilled in the art of protein expression.

Similarly useful as host cells suitable for the present invention arebacterial cells. For example, the various strains of E. coli (e.g.,HB101, DH5α, DH10, and MC1061) are well-known as host cells in the fieldof biotechnology. Various strains of B. subtilis, Pseudomonas spp.,other Bacillus spp., Streptomyces spp., and the like may also beemployed in this method.

Many strains of yeast cells known to those skilled in the art are alsoavailable as host cells for the expression of the polypeptides of thepresent invention. Preferred yeast cells include, for example,Saccharomyces cerivisae and Pichia pastoris.

Additionally, where desired, insect cell systems may be utilized in themethods of the present invention. Such systems are described, forexample, in Kitts et al., 1993, Biotechniques, 14:810-17; Lucklow, 1993,Curr. Opin. Biotechnol. 4:564-72; and Lucklow et al., 1993, J. Virol.,67:4566-79. Preferred insect cells are Sf-9 and Hi5 (Invitrogen).

One may also use transgenic animals to express glycosylated IFN-Lpolypeptides. For example, one may use a transgenic milk-producinganimal (a cow or goat, for example) and obtain the present glycosylatedpolypeptide in the animal milk. One may also use plants to produce IFN-Lpolypeptides, however, in general, the glycosylation occurring in plantsis different from that produced in mammalian cells, and may result in aglycosylated product which is not suitable for human therapeutic use.

Polypeptide Production

Host cells comprising an IFN-L polypeptide expression vector may becultured using standard media well known to the skilled artisan. Themedia will usually contain all nutrients necessary for the growth andsurvival of the cells. Suitable media for culturing E. coli cellsinclude, for example, Luria Broth (LB) and/or Terrific Broth (TB).Suitable media for culturing eukaryotic cells include Roswell ParkMemorial Institute medium 1640 (RPMI 1640), Minimal Essential Medium(MEM) and/or Dulbecco's Modified Eagle Medium (DMEM), all of which maybe supplemented with serum and/or growth factors as necessary for theparticular cell line being cultured. A suitable medium for insectcultures is Grace's medium supplemented with yeastolate, lactalbuminhydrolysate, and/or fetal calf serum as necessary.

Typically, an antibiotic or other compound useful for selective growthof transfected or transformed cells is added as a supplement to themedia. The compound to be used will be dictated by the selectable markerelement present on the plasmid with which the host cell was transformed.For example, where the selectable marker element is kanamycinresistance, the compound added to the culture medium will be kanamycin.Other compounds for selective growth include ampicillin, tetracycline,and neomycin.

The amount of an IFN-L polypeptide produced by a host cell can beevaluated using standard methods known in the art. Such methods include,without limitation, Western blot analysis, SDS-polyacrylamide gelelectrophoresis, non-denaturing gel electrophoresis, High PerformanceLiquid Chromatography (HPLC) separation, immunoprecipitation, and/oractivity assays such as DNA binding gel shift assays.

If an IFN-L polypeptide has been designed to be secreted from the hostcells, the majority of polypeptide may be found in the cell culturemedium. If however, the IFN-L polypeptide is not secreted from the hostcells, it will be present in the cytoplasm and/or the nucleus (foreukaryotic host cells) or in the cytosol (for gram-negative bacteriahost cells).

For an IFN-L polypeptide situated in the host cell cytoplasm and/ornucleus (for eukaryotic host cells) or in the cytosol (for bacterialhost cells), the intracellular material (including inclusion bodies forgram-negative bacteria) can be extracted from the host cell using anystandard technique known to the skilled artisan. For example, the hostcells can be lysed to release the contents of the periplasm/cytoplasm byFrench press, homogenization, and/or sonication followed bycentrifugation.

If an IFN-L polypeptide has formed inclusion bodies in the cytosol, theinclusion bodies can often bind to the inner and/or outer cellularmembranes and thus will be found primarily in the pellet material aftercentrifugation. The pellet material can then be treated at pH extremesor with a chaotropic agent such as a detergent, guanidine, guanidinederivatives, urea, or urea derivatives in the presence of a reducingagent such as dithiothreitol at alkaline pH or tris carboxyethylphosphine at acid pH to release, break apart, and solubilize theinclusion bodies. The solubilized IFN-L polypeptide can then be analyzedusing gel electrophoresis, immunoprecipitation, or the like. If it isdesired to isolate the IFN-L polypeptide, isolation may be accomplishedusing standard methods such as those described herein and in Marston etal., 1990, Meth. Enz., 182:264-75.

In some cases, an IFN-L polypeptide may not be biologically active uponisolation. Various methods for “refolding” or converting the polypeptideto its tertiary structure and generating disulfide linkages can be usedto restore biological activity. Such methods include exposing thesolubilized polypeptide to a pH usually above 7 and in the presence of aparticular concentration of a chaotrope. The selection of chaotrope isvery similar to the choices used for inclusion body solubilization, butusually the chaotrope is used at a lower concentration and is notnecessarily the same as chaotropes used for the solubilization. In mostcases the refolding/oxidation solution will also contain a reducingagent or the reducing agent plus its oxidized form in a specific ratioto generate a particular redox potential allowing for disulfideshuffling to occur in the formation of the protein's cysteine bridges.Some of the commonly used redox couples include cysteine/cystamine,glutathione (GSH)/dithiobis GSH, cupric chloride,dithiothreitol(DTT)/dithiane DTT, and2-2-mercaptoethanol(bME)/dithio-b(ME). In many instances, a cosolventmay be used or may be needed to increase the efficiency of therefolding, and the more common reagents used for this purpose includeglycerol, polyethylene glycol of various molecular weights, arginine andthe like.

If inclusion bodies are not formed to a significant degree uponexpression of an IFN-L polypeptide, then the polypeptide will be foundprimarily in the supernatant after centrifugation of the cellhomogenate. The polypeptide may be further isolated from the supernatantusing methods such as those described herein.

The purification of an IFN-L polypeptide from solution can beaccomplished using a variety of techniques. If the polypeptide has beensynthesized such that it contains a tag such as Hexahistidine (IFN-Lpolypeptide/hexaHis) or other small peptide such as FLAG (Eastman KodakCo., New Haven, Conn.) or myc (Invitrogen, Carlsbad, Calif.) at eitherits carboxyl- or amino-terminus, it may be purified in a one-stepprocess by passing the solution through an affinity column where thecolumn matrix has a high affinity for the tag.

For example, polyhistidine binds with great affinity and specificity tonickel. Thus, an affinity column of nickel (such as the Qiagen® nickelcolumns) can be used for purification of IFN-L polypeptide/polyHis. See,e.g., Current Protocols in Molecular Biology §10.11.8 (Ausubel et al.,eds., Green Publishers Inc. and Wiley and Sons 1993).

Additionally, IFN-L polypeptides may be purified through the use of amonoclonal antibody that is capable of specifically recognizing andbinding to an IFN-L polypeptide.

Other suitable procedures for purification include, without limitation,affinity chromatography, immunoaffinity chromatography, ion exchangechromatography, molecular sieve chromatography, HPLC, electrophoresis(including native gel electrophoresis) followed by gel elution, andpreparative isoelectric focusing (“Isoprime” machine/technique, HoeferScientific, San Francisco, Calif.). In some cases, two or morepurification techniques may be combined to achieve increased purity.

IFN-L polypeptides may also be prepared by chemical synthesis methods(such as solid phase peptide synthesis) using techniques known in theart such as those set forth by Merrifield et al., 1963, J. Am. Chem.Soc. 85:2149; Houghten et al., 1985, Proc Natl Acad. Sci. USA 82:5132;and Stewart and Young, Solid Phase Peptide Synthesis (Pierce ChemicalCo. 1984). Such polypeptides may be synthesized with or without amethionine on the amino-terminus Chemically synthesized IFN-Lpolypeptides may be oxidized using methods set forth in these referencesto form disulfide bridges. Chemically synthesized IFN-L polypeptides areexpected to have comparable biological activity to the correspondingIFN-L polypeptides produced recombinantly or purified from naturalsources, and thus may be used interchangeably with a recombinant ornatural IFN-L polypeptide.

Another means of obtaining IFN-L polypeptide is via purification frombiological samples such as source tissues and/or fluids in which theIFN-L polypeptide is naturally found. Such purification can be conductedusing methods for protein purification as described herein. The presenceof the IFN-L polypeptide during purification may be monitored, forexample, using an antibody prepared against recombinantly produced IFN-Lpolypeptide or peptide fragments thereof.

A number of additional methods for producing nucleic acids andpolypeptides are known in the art, and the methods can be used toproduce polypeptides having specificity for IFN-L polypeptide. See,e.g., Roberts et al., 1997, Proc. Natl. Acad. Sci. U.S.A. 94:12297-303,which describes the production of fusion proteins between an mRNA andits encoded peptide. See also, Roberts, 1999, Curr. Opin. Chem. Biol.3:268-73. Additionally, U.S. Pat. No. 5,824,469 describes methods forobtaining oligonucleotides capable of carrying out a specific biologicalfunction. The procedure involves generating a heterogeneous pool ofoligonucleotides, each having a 5′ randomized sequence, a centralpreselected sequence, and a 3′ randomized sequence. The resultingheterogeneous pool is introduced into a population of cells that do notexhibit the desired biological function. Subpopulations of the cells arethen screened for those that exhibit a predetermined biologicalfunction. From that subpopulation, oligonucleotides capable of carryingout the desired biological function are isolated.

U.S. Pat. Nos. 5,763,192; 5,814,476; 5,723,323; and 5,817,483 describeprocesses for producing peptides or polypeptides. This is done byproducing stochastic genes or fragments thereof, and then introducingthese genes into host cells which produce one or more proteins encodedby the stochastic genes. The host cells are then screened to identifythose clones producing peptides or polypeptides having the desiredactivity.

Another method for producing peptides or polypeptides is described inPCT/US98/20094 (WO99/15650) filed by Athersys, Inc. Known as “RandomActivation of Gene Expression for Gene Discovery” (RAGE-GD), the processinvolves the activation of endogenous gene expression or over-expressionof a gene by in situ recombination methods. For example, expression ofan endogenous gene is activated or increased by integrating a regulatorysequence into the target cell which is capable of activating expressionof the gene by non-homologous or illegitimate recombination. The targetDNA is first subjected to radiation, and a genetic promoter inserted.The promoter eventually locates a break at the front of a gene,initiating transcription of the gene. This results in expression of thedesired peptide or polypeptide.

It will be appreciated that these methods can also be used to createcomprehensive IFN-L polypeptide expression libraries, which cansubsequently be used for high throughput phenotypic screening in avariety of assays, such as biochemical assays, cellular assays, andwhole organism assays (e.g., plant, mouse, etc.).

Synthesis

It will be appreciated by those skilled in the art that the nucleic acidand polypeptide molecules described herein may be produced byrecombinant and other means.

Selective Binding Agents

The term “selective binding agent” refers to a molecule that hasspecificity for one or more IFN-L polypeptides. Suitable selectivebinding agents include, but are not limited to, antibodies andderivatives thereof, polypeptides, and small molecules. Suitableselective binding agents may be prepared using methods known in the art.An exemplary IFN-L polypeptide selective binding agent of the presentinvention is capable of binding a certain portion of the IFN-Lpolypeptide thereby inhibiting the binding of the polypeptide to anIFN-L polypeptide receptor.

Selective binding agents such as antibodies and antibody fragments thatbind IFN-L polypeptides are within the scope of the present invention.The antibodies may be polyclonal including monospecific polyclonal;monoclonal (MAbs); recombinant; chimeric; humanized, such asCDR-grafted; human; single chain; and/or bispecific; as well asfragments; variants; or derivatives thereof. Antibody fragments includethose portions of the antibody that bind to an epitope on the IFN-Lpolypeptide. Examples of such fragments include Fab and F(ab′) fragmentsgenerated by enzymatic cleavage of full-length antibodies. Other bindingfragments include those generated by recombinant DNA techniques, such asthe expression of recombinant plasmids containing nucleic acid sequencesencoding antibody variable regions.

Polyclonal antibodies directed toward an IFN-L polypeptide generally areproduced in animals (e.g., rabbits or mice) by means of multiplesubcutaneous or intraperitoneal injections of IFN-L polypeptide and anadjuvant. It may be useful to conjugate an IFN-L polypeptide to acarrier protein that is immunogenic in the species to be immunized, suchas keyhole limpet hemocyanin, serum, albumin, bovine thyroglobulin, orsoybean trypsin inhibitor. Also, aggregating agents such as alum areused to enhance the immune response. After immunization, the animals arebled and the serum is assayed for anti-IFN-L antibody titer.

Monoclonal antibodies directed toward IFN-L polypeptides are producedusing any method that provides for the production of antibody moleculesby continuous cell lines in culture. Examples of suitable methods forpreparing monoclonal antibodies include the hybridoma methods of Kohleret al., 1975, Nature 256:495-97 and the human B-cell hybridoma method(Kozbor, 1984, J. Immunol. 133:3001; Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications 51-63 (Marcel Dekker, Inc.,1987). Also provided by the invention are hybridoma cell lines thatproduce monoclonal antibodies reactive with IFN-L polypeptides.

Monoclonal antibodies of the invention may be modified for use astherapeutics. One embodiment is a “chimeric” antibody in which a portionof the heavy (H) and/or light (L) chain is identical with or homologousto a corresponding sequence in antibodies derived from a particularspecies or belonging to a particular antibody class or subclass, whilethe remainder of the chain(s) is/are identical with or homologous to acorresponding sequence in antibodies derived from another species orbelonging to another antibody class or subclass. Also included arefragments of such antibodies, so long as they exhibit the desiredbiological activity. See U.S. Pat. No. 4,816,567; Morrison et al., 1985,Proc. Natl. Acad. Sci. 81:6851-55.

In another embodiment, a monoclonal antibody of the invention is a“humanized” antibody. Methods for humanizing non-human antibodies arewell known in the art. See U.S. Pat. Nos. 5,585,089 and 5,693,762.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source that is non-human. Humanization can beperformed, for example, using methods described in the art (Jones etal., 1986, Nature 321:522-25; Riechmann et al., 1998, Nature 332:323-27;Verhoeyen et al., 1988, Science 239:1534-36), by substituting at least aportion of a rodent complementarity-determining region (CDR) for thecorresponding regions of a human antibody.

Also encompassed by the invention are human antibodies that bind IFN-Lpolypeptides. Using transgenic animals (e.g., mice) that are capable ofproducing a repertoire of human antibodies in the absence of endogenousimmunoglobulin production such antibodies are produced by immunizationwith an IFN-L polypeptide antigen (i.e., having at least 6 contiguousamino acids), optionally conjugated to a carrier. See, e.g., Jakobovitset al., 1993, Proc. Natl. Acad. Sci. 90:2551-55; Jakobovits et al.,1993, Nature 362:255-58; Bruggermann et al., 1993, Year in Immuno. 7:33.In one method, such transgenic animals are produced by incapacitatingthe endogenous loci encoding the heavy and light immunoglobulin chainstherein, and inserting loci encoding human heavy and light chainproteins into the genome thereof. Partially modified animals, that isthose having less than the full complement of modifications, are thencross-bred to obtain an animal having all of the desired immune systemmodifications. When administered an immunogen, these transgenic animalsproduce antibodies with human (rather than, e.g., murine) amino acidsequences, including variable regions which are immunospecific for theseantigens. See PCT App. Nos. PCT/US96/05928 and PCT/US93/06926.Additional methods are described in U.S. Pat. No. 5,545,807, PCT App.Nos. PCT/US91/245 and PCT/GB89/01207, and in European Patent Nos.546073B1 and 546073A1. Human antibodies can also be produced by theexpression of recombinant DNA in host cells or by expression inhybridoma cells as described herein.

In an alternative embodiment, human antibodies can also be produced fromphage-display libraries (Hoogenboom et al., 1991, J. Mol. Biol. 227:381;Marks et al., 1991, J. Mol. Biol. 222:581). These processes mimic immuneselection through the display of antibody repertoires on the surface offilamentous bacteriophage, and subsequent selection of phage by theirbinding to an antigen of choice. One such technique is described in PCTApp. No. PCT/US98/17364, which describes the isolation of high affinityand functional agonistic antibodies for MPL- and msk-receptors usingsuch an approach.

Chimeric, CDR grafted, and humanized antibodies are typically producedby recombinant methods. Nucleic acids encoding the antibodies areintroduced into host cells and expressed using materials and proceduresdescribed herein. In a preferred embodiment, the antibodies are producedin mammalian host cells, such as CHO cells. Monoclonal (e.g., human)antibodies may be produced by the expression of recombinant DNA in hostcells or by expression in hybridoma cells as described herein.

The anti-IFN-L antibodies of the invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays (Sola, MonoclonalAntibodies: A Manual of Techniques 147-158 (CRC Press, Inc., 1987)) forthe detection and quantitation of IFN-L polypeptides. The antibodieswill bind IFN-L polypeptides with an affinity that is appropriate forthe assay method being employed.

For diagnostic applications, in certain embodiments, anti-IFN-Lantibodies may be labeled with a detectable moiety. The detectablemoiety can be any one that is capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, ¹²⁵I, ⁹⁹Tc, ¹¹¹In, or⁶⁷Ga; a fluorescent or chemiluminescent compound, such as fluoresceinisothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkalinephosphatase, β-galactosidase, or horseradish peroxidase (Bayer, et al.,1990, Meth. Enz. 184:138-63).

Competitive binding assays rely on the ability of a labeled standard(e.g., an IFN-L polypeptide, or an immunologically reactive portionthereof) to compete with the test sample analyte (an IFN-L polypeptide)for binding with a limited amount of anti-IFN-L antibody. The amount ofan IFN-L polypeptide in the test sample is inversely proportional to theamount of standard that becomes bound to the antibodies. To facilitatedetermining the amount of standard that becomes bound, the antibodiestypically are insolubilized before or after the competition, so that thestandard and analyte that are bound to the antibodies may convenientlybe separated from the standard and analyte which remain unbound.

Sandwich assays typically involve the use of two antibodies, eachcapable of binding to a different immunogenic portion, or epitope, ofthe protein to be detected and/or quantitated. In a sandwich assay, thetest sample analyte is typically bound by a first antibody which isimmobilized on a solid support, and thereafter a second antibody bindsto the analyte, thus forming an insoluble three-part complex. See, e.g.,U.S. Pat. No. 4,376,110. The second antibody may itself be labeled witha detectable moiety (direct sandwich assays) or may be measured using ananti-immunoglobulin antibody that is labeled with a detectable moiety(indirect sandwich assays). For example, one type of sandwich assay isan enzyme-linked immunosorbent assay (ELISA), in which case thedetectable moiety is an enzyme.

The selective binding agents, including anti-IFN-L antibodies, are alsouseful for in vivo imaging. An antibody labeled with a detectable moietymay be administered to an animal, preferably into the bloodstream, andthe presence and location of the labeled antibody in the host assayed.The antibody may be labeled with any moiety that is detectable in ananimal, whether by nuclear magnetic resonance, radiology, or otherdetection means known in the art.

Selective binding agents of the invention, including antibodies, may beused as therapeutics. These therapeutic agents are generally agonists orantagonists, in that they either enhance or reduce, respectively, atleast one of the biological activities of an IFN-L polypeptide. In oneembodiment, antagonist antibodies of the invention are antibodies orbinding fragments thereof which are capable of specifically binding toan IFN-L polypeptide and which are capable of inhibiting or eliminatingthe functional activity of an IFN-L polypeptide in vivo or in vitro. Inpreferred embodiments, the selective binding agent, e.g., an antagonistantibody, will inhibit the functional activity of an IFN-L polypeptideby at least about 50%, and preferably by at least about 80%. In anotherembodiment, the selective binding agent may be an anti-IFN-L polypeptideantibody that is capable of interacting with an IFN-L polypeptidebinding partner (a ligand or receptor) thereby inhibiting or eliminatingIFN-L polypeptide activity in vitro or in vivo. Selective bindingagents, including agonist and antagonist anti-IFN-L polypeptideantibodies, are identified by screening assays that are well known inthe art.

The invention also relates to a kit comprising IFN-L selective bindingagents (such as antibodies) and other reagents useful for detectingIFN-L polypeptide levels in biological samples. Such reagents mayinclude a detectable label, blocking serum, positive and negativecontrol samples, and detection reagents.

Microarrays

It will be appreciated that DNA microarray technology can be utilized inaccordance with the present invention. DNA microarrays are miniature,high-density arrays of nucleic acids positioned on a solid support, suchas glass. Each cell or element within the array contains numerous copiesof a single nucleic acid species that acts as a target for hybridizationwith a complementary nucleic acid sequence (e.g., mRNA). In expressionprofiling using DNA microarray technology, mRNA is first extracted froma cell or tissue sample and then converted enzymatically tofluorescently labeled cDNA. This material is hybridized to themicroarray and unbound cDNA is removed by washing. The expression ofdiscrete genes represented on the array is then visualized byquantitating the amount of labeled cDNA that is specifically bound toeach target nucleic acid molecule. In this way, the expression ofthousands of genes can be quantitated in a high throughput, parallelmanner from a single sample of biological material.

This high throughput expression profiling has a broad range ofapplications with respect to the IFN-L molecules of the invention,including, but not limited to: the identification and validation ofIFN-L disease-related genes as targets for therapeutics; moleculartoxicology of related IFN-L molecules and inhibitors thereof;stratification of populations and generation of surrogate markers forclinical trials; and enhancing related IFN-L polypeptide small moleculedrug discovery by aiding in the identification of selective compounds inhigh throughput screens.

Chemical Derivatives

Chemically modified derivatives of IFN-L polypeptides may be prepared byone skilled in the art, given the disclosures described herein. IFN-Lpolypeptide derivatives are modified in a manner that isdifferent—either in the type or location of the molecules naturallyattached to the polypeptide. Derivatives may include molecules formed bythe deletion of one or more naturally-attached chemical groups. Thepolypeptide comprising the amino acid sequence of either SEQ ID NO: 2 orSEQ ID NO: 5, or other IFN-L polypeptide, may be modified by thecovalent attachment of one or more polymers. For example, the polymerselected is typically water-soluble so that the protein to which it isattached does not precipitate in an aqueous environment, such as aphysiological environment. Included within the scope of suitablepolymers is a mixture of polymers. Preferably, for therapeutic use ofthe end-product preparation, the polymer will be pharmaceuticallyacceptable.

The polymers each may be of any molecular weight and may be branched orunbranched. The polymers each typically have an average molecular weightof between about 2 kDa to about 100 kDa (the term “about” indicatingthat in preparations of a water-soluble polymer, some molecules willweigh more, some less, than the stated molecular weight). The averagemolecular weight of each polymer is preferably between about 5 kDa andabout 50 kDa, more preferably between about 12 kDa and about 40 kDa andmost preferably between about 20 kDa and about 35 kDa.

Suitable water-soluble polymers or mixtures thereof include, but are notlimited to, N-linked or O-linked carbohydrates, sugars, phosphates,polyethylene glycol (PEG) (including the forms of PEG that have beenused to derivatize proteins, including mono-(C₁-C₁₀), alkoxy-, oraryloxy-polyethylene glycol), monomethoxy-polyethylene glycol, dextran(such as low molecular weight dextran of, for example, about 6 kD),cellulose, or other carbohydrate based polymers, poly-(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers,polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols(e.g., glycerol), and polyvinyl alcohol. Also encompassed by the presentinvention are bifunctional crosslinking molecules which may be used toprepare covalently attached IFN-L polypeptide multimers.

In general, chemical derivatization may be performed under any suitablecondition used to react a protein with an activated polymer molecule.Methods for preparing chemical derivatives of polypeptides willgenerally comprise the steps of: (a) reacting the polypeptide with theactivated polymer molecule (such as a reactive ester or aldehydederivative of the polymer molecule) under conditions whereby thepolypeptide comprising the amino acid sequence of either SEQ ID NO: 2 orSEQ ID NO: 5, or other IFN-L polypeptide, becomes attached to one ormore polymer molecules, and (b) obtaining the reaction products. Theoptimal reaction conditions will be determined based on known parametersand the desired result. For example, the larger the ratio of polymermolecules to protein, the greater the percentage of attached polymermolecule. In one embodiment, the IFN-L polypeptide derivative may have asingle polymer molecule moiety at the amino-terminus. See, e.g. U.S.Pat. No. 5,234,784.

The pegylation of a polypeptide may be specifically carried out usingany of the pegylation reactions known in the art. Such reactions aredescribed, for example, in the following references: Francis et al.,1992, Focus on Growth Factors 3:4-10; European Patent Nos. 0154316 and0401384; and U.S. Pat. No. 4,179,337. For example, pegylation may becarried out via an acylation reaction or an alkylation reaction with areactive polyethylene glycol molecule (or an analogous reactivewater-soluble polymer) as described herein. For the acylation reactions,a selected polymer should have a single reactive ester group. Forreductive alkylation, a selected polymer should have a single reactivealdehyde group. A reactive aldehyde is, for example, polyethylene glycolpropionaldehyde, which is water stable, or mono C₁-C₁₀ alkoxy or aryloxyderivatives thereof (see U.S. Pat. No. 5,252,714).

In another embodiment, IFN-L polypeptides may be chemically coupled tobiotin. The biotin/IFN-L polypeptide molecules are then allowed to bindto avidin, resulting in tetravalent avidin/biotin/IFN-L polypeptidemolecules. IFN-L polypeptides may also be covalently coupled todinitrophenol (DNP) or trinitrophenol (TNP) and the resulting conjugatesprecipitated with anti-DNP or anti-TNP-IgM to form decameric conjugateswith a valency of 10.

Generally, conditions that may be alleviated or modulated by theadministration of the present IFN-L polypeptide derivatives includethose described herein for IFN-L polypeptides. However, the IFN-Lpolypeptide derivatives disclosed herein may have additional activities,enhanced or reduced biological activity, or other characteristics, suchas increased or decreased half-life, as compared to the non-derivatizedmolecules.

Genetically Engineered Non-Human Animals

Additionally included within the scope of the present invention arenon-human animals such as mice, rats, or other rodents; rabbits, goats,sheep, or other farm animals, in which the genes encoding native IFN-Lpolypeptide have been disrupted (i.e., “knocked out”) such that thelevel of expression of IFN-L polypeptide is significantly decreased orcompletely abolished. Such animals may be prepared using techniques andmethods such as those described in U.S. Pat. No. 5,557,032.

The present invention further includes non-human animals such as mice,rats, or other rodents; rabbits, goats, sheep, or other farm animals, inwhich either the native form of an IFN-L gene for that animal or aheterologous IFN-L gene is over-expressed by the animal, therebycreating a “transgenic” animal. Such transgenic animals may be preparedusing well known methods such as those described in U.S. Pat. No.5,489,743 and PCT Pub. No. WO 94/28122.

The present invention further includes non-human animals in which thepromoter for one or more of the IFN-L polypeptides of the presentinvention is either activated or inactivated (e.g., by using homologousrecombination methods) to alter the level of expression of one or moreof the native IFN-L polypeptides.

These non-human animals may be used for drug candidate screening. Insuch screening, the impact of a drug candidate on the animal may bemeasured. For example, drug candidates may decrease or increase theexpression of the IFN-L gene. In certain embodiments, the amount ofIFN-L polypeptide that is produced may be measured after the exposure ofthe animal to the drug candidate. Additionally, in certain embodiments,one may detect the actual impact of the drug candidate on the animal.For example, over-expression of a particular gene may result in, or beassociated with, a disease or pathological condition. In such cases, onemay test a drug candidate's ability to decrease expression of the geneor its ability to prevent or inhibit a pathological condition. In otherexamples, the production of a particular metabolic product such as afragment of a polypeptide, may result in, or be associated with, adisease or pathological condition. In such cases, one may test a drugcandidate's ability to decrease the production of such a metabolicproduct or its ability to prevent or inhibit a pathological condition.

Assaying for Other Modulators of IFN-L Polypeptide Activity

In some situations, it may be desirable to identify molecules that aremodulators, i.e., agonists or antagonists, of the activity of IFN-Lpolypeptide. Natural or synthetic molecules that modulate IFN-Lpolypeptide may be identified using one or more screening assays, suchas those described herein. Such molecules may be administered either inan ex vivo manner or in an in vivo manner by injection, or by oraldelivery, implantation device, or the like.

“Test molecule” refers to a molecule that is under evaluation for theability to modulate (i.e., increase or decrease) the activity of anIFN-L polypeptide. Most commonly, a test molecule will interact directlywith an IFN-L polypeptide. However, it is also contemplated that a testmolecule may also modulate IFN-L polypeptide activity indirectly, suchas by affecting IFN-L gene expression, or by binding to an IFN-Lpolypeptide binding partner (e.g., receptor or ligand). In oneembodiment, a test molecule will bind to an IFN-L polypeptide with anaffinity constant of at least about 10⁻⁶ M, preferably about 10⁻⁸ M,more preferably about 10⁻⁹ M, and even more preferably about 10⁻¹⁰ M.

Methods for identifying compounds that interact with IFN-L polypeptidesare encompassed by the present invention. In certain embodiments, anIFN-L polypeptide is incubated with a test molecule under conditionsthat permit the interaction of the test molecule with an IFN-Lpolypeptide, and the extent of the interaction is measured. The testmolecule can be screened in a substantially purified form or in a crudemixture.

In certain embodiments, an IFN-L polypeptide agonist or antagonist maybe a protein, peptide, carbohydrate, lipid, or small molecular weightmolecule that interacts with IFN-L polypeptide to regulate its activity.Molecules which regulate IFN-L polypeptide expression include nucleicacids which are complementary to nucleic acids encoding an IFN-Lpolypeptide, or are complementary to nucleic acids sequences whichdirect or control the expression of IFN-L polypeptide, and which act asanti-sense regulators of expression.

Once a test molecule has been identified as interacting with an IFN-Lpolypeptide, the molecule may be further evaluated for its ability toincrease or decrease IFN-L polypeptide activity. The measurement of theinteraction of a test molecule with IFN-L polypeptide may be carried outin several formats, including cell-based binding assays, membranebinding assays, solution-phase assays, and immunoassays. In general, atest molecule is incubated with an IFN-L polypeptide for a specifiedperiod of time, and IFN-L polypeptide activity is determined by one ormore assays for measuring biological activity.

The interaction of test molecules with IFN-L polypeptides may also beassayed directly using polyclonal or monoclonal antibodies in animmunoassay. Alternatively, modified forms of IFN-L polypeptidescontaining epitope tags as described herein may be used in solution andimmunoassays.

In the event that IFN-L polypeptides display biological activity throughan interaction with a binding partner (e.g., a receptor or a ligand), avariety of in vitro assays may be used to measure the binding of anIFN-L polypeptide to the corresponding binding partner (such as aselective binding agent, receptor, or ligand). These assays may be usedto screen test molecules for their ability to increase or decrease therate and/or the extent of binding of an IFN-L polypeptide to its bindingpartner. In one assay, an IFN-L polypeptide is immobilized in the wellsof a microtiter plate. Radiolabeled IFN-L polypeptide binding partner(for example, iodinated IFN-L polypeptide binding partner) and a testmolecule can then be added either one at a time (in either order) orsimultaneously to the wells. After incubation, the wells can be washedand counted for radioactivity, using a scintillation counter, todetermine the extent to which the binding partner bound to the IFN-Lpolypeptide. Typically, a molecule will be tested over a range ofconcentrations, and a series of control wells lacking one or moreelements of the test assays can be used for accuracy in the evaluationof the results. An alternative to this method involves reversing the“positions” of the proteins, i.e., immobilizing IFN-L polypeptidebinding partner to the microtiter plate wells, incubating with the testmolecule and radiolabeled IFN-L polypeptide, and determining the extentof IFN-L polypeptide binding. See, e.g., Current Protocols in MolecularBiology, chap. 18 (Ausubel et al., eds., Green Publishers Inc. and Wileyand Sons 1995).

As an alternative to radiolabeling, an IFN-L polypeptide or its bindingpartner may be conjugated to biotin, and the presence of biotinylatedprotein can then be detected using streptavidin linked to an enzyme,such as horse radish peroxidase (HRP) or alkaline phosphatase (AP),which can be detected colorometrically, or by fluorescent tagging ofstreptavidin. An antibody directed to an IFN-L polypeptide or to anIFN-L polypeptide binding partner, and which is conjugated to biotin,may also be used for purposes of detection following incubation of thecomplex with enzyme-linked streptavidin linked to AP or HRP.

A IFN-L polypeptide or an IFN-L polypeptide binding partner can also beimmobilized by attachment to agarose beads, acrylic beads, or othertypes of such inert solid phase substrates. The substrate-proteincomplex can be placed in a solution containing the complementary proteinand the test compound. After incubation, the beads can be precipitatedby centrifugation, and the amount of binding between an IFN-Lpolypeptide and its binding partner can be assessed using the methodsdescribed herein. Alternatively, the substrate-protein complex can beimmobilized in a column with the test molecule and complementary proteinpassing through the column. The formation of a complex between an IFN-Lpolypeptide and its binding partner can then be assessed using any ofthe techniques described herein (e.g., radiolabelling or antibodybinding).

Another in vitro assay that is useful for identifying a test moleculewhich increases or decreases the formation of a complex between an IFN-Lpolypeptide binding protein and an IFN-L polypeptide binding partner isa surface plasmon resonance detector system such as the BIAcore assaysystem (Pharmacia, Piscataway, N.J.). The BIAcore system is utilized asspecified by the manufacturer. This assay essentially involves thecovalent binding of either IFN-L polypeptide or an IFN-L polypeptidebinding partner to a dextran-coated sensor chip that is located in adetector. The test compound and the other complementary protein can thenbe injected, either simultaneously or sequentially, into the chambercontaining the sensor chip. The amount of complementary protein thatbinds can be assessed based on the change in molecular mass that isphysically associated with the dextran-coated side of the sensor chip,with the change in molecular mass being measured by the detector system.

In some cases, it may be desirable to evaluate two or more testcompounds together for their ability to increase or decrease theformation of a complex between an IFN-L polypeptide and an IFN-Lpolypeptide binding partner. In these cases, the assays set forth hereincan be readily modified by adding such additional test compound(s)either simultaneously with, or subsequent to, the first test compound.The remainder of the steps in the assay are as set forth herein.

In vitro assays such as those described herein may be usedadvantageously to screen large numbers of compounds for an effect on theformation of a complex between an IFN-L polypeptide and IFN-Lpolypeptide binding partner. The assays may be automated to screencompounds generated in phage display, synthetic peptide, and chemicalsynthesis libraries.

Compounds which increase or decrease the formation of a complex betweenan IFN-L polypeptide and an IFN-L polypeptide binding partner may alsobe screened in cell culture using cells and cell lines expressing eitherIFN-L polypeptide or IFN-L polypeptide binding partner. Cells and celllines may be obtained from any mammal, but preferably will be from humanor other primate, canine, or rodent sources. The binding of an IFN-Lpolypeptide to cells expressing IFN-L polypeptide binding partner at thesurface is evaluated in the presence or absence of test molecules, andthe extent of binding may be determined by, for example, flow cytometryusing a biotinylated antibody to an IFN-L polypeptide binding partner.Cell culture assays can be used advantageously to further evaluatecompounds that score positive in protein binding assays describedherein.

Cell cultures can also be used to screen the impact of a drug candidate.For example, drug candidates may decrease or increase the expression ofthe IFN-L gene. In certain embodiments, the amount of IFN-L polypeptideor an IFN-L polypeptide fragment that is produced may be measured afterexposure of the cell culture to the drug candidate. In certainembodiments, one may detect the actual impact of the drug candidate onthe cell culture. For example, the over-expression of a particular genemay have a particular impact on the cell culture. In such cases, one maytest a drug candidate's ability to increase or decrease the expressionof the gene or its ability to prevent or inhibit a particular impact onthe cell culture. In other examples, the production of a particularmetabolic product such as a fragment of a polypeptide, may result in, orbe associated with, a disease or pathological condition. In such cases,one may test a drug candidate's ability to decrease the production ofsuch a metabolic product in a cell culture.

Internalizing Proteins

The tat protein sequence (from HIV) can be used to internalize proteinsinto a cell. See, e.g., Falwell et al., 1994, Proc. Natl. Acad. Sci.U.S.A. 91:664-68. For example, an 11 amino acid sequence(Y-G-R-K-K-R-R-Q-R-R-R; SEQ ID NO: 18) of the HIV tat protein (termedthe “protein transduction domain,” or TAT PDT) has been described asmediating delivery across the cytoplasmic membrane and the nuclearmembrane of a cell. See Schwarze et al., 1999, Science 285:1569-72; andNagahara et al., 1998, Nat. Med. 4:1449-52. In these procedures,FITC-constructs (FITC-labeled G-G-G-G-Y-G-R-K-K-R-R-Q-R-R-R; SEQ ID NO:19), which penetrate tissues following intraperitoneal administration,are prepared, and the binding of such constructs to cells is detected byfluorescence-activated cell sorting (FACS) analysis. Cells treated witha tat-β-gal fusion protein will demonstrate β-gal activity. Followinginjection, expression of such a construct can be detected in a number oftissues, including liver, kidney, lung, heart, and brain tissue. It isbelieved that such constructs undergo some degree of unfolding in orderto enter the cell, and as such, may require a refolding following entryinto the cell.

It will thus be appreciated that the tat protein sequence may be used tointernalize a desired polypeptide into a cell. For example, using thetat protein sequence, an IFN-L antagonist (such as an anti-IFN-Lselective binding agent, small molecule, soluble receptor, or antisenseoligonucleotide) can be administered intracellularly to inhibit theactivity of an IFN-L molecule. As used herein, the term “IFN-L molecule”refers to both IFN-L nucleic acid molecules and IFN-L polypeptides asdefined herein. Where desired, the IFN-L protein itself may also beinternally administered to a cell using these procedures. See also,Straus, 1999, Science 285:1466-67.

Cell Source Identification Using IFN-L Polypeptide

In accordance with certain embodiments of the invention, it may beuseful to be able to determine the source of a certain cell typeassociated with an IFN-L polypeptide. For example, it may be useful todetermine the origin of a disease or pathological condition as an aid inselecting an appropriate therapy. In certain embodiments, nucleic acidsencoding an IFN-L polypeptide can be used as a probe to identify cellsdescribed herein by screening the nucleic acids of the cells with such aprobe. In other embodiments, one may use anti-IFN-L polypeptideantibodies to test for the presence of IFN-L polypeptide in cells, andthus, determine if such cells are of the types described herein.

IFN-L Polypeptide Compositions and Administration

Therapeutic compositions are within the scope of the present invention.Such IFN-L polypeptide pharmaceutical compositions may comprise atherapeutically effective amount of an IFN-L polypeptide or an IFN-Lnucleic acid molecule in admixture with a pharmaceutically orphysiologically acceptable formulation agent selected for suitabilitywith the mode of administration. Pharmaceutical compositions maycomprise a therapeutically effective amount of one or more IFN-Lpolypeptide selective binding agents in admixture with apharmaceutically or physiologically acceptable formulation agentselected for suitability with the mode of administration.

Acceptable formulation materials preferably are nontoxic to recipientsat the dosages and concentrations employed.

The pharmaceutical composition may contain formulation materials formodifying, maintaining, or preserving, for example, the pH, osmolarity,viscosity, clarity, color, isotonicity, odor, sterility, stability, rateof dissolution or release, adsorption, or penetration of thecomposition. Suitable formulation materials include, but are not limitedto, amino acids (such as glycine, glutamine, asparagine, arginine, orlysine), antimicrobials, antioxidants (such as ascorbic acid, sodiumsulfite, or sodium hydrogen-sulfite), buffers (such as borate,bicarbonate, Tris-HCl, citrates, phosphates, or other organic acids),bulking agents (such as mannitol or glycine), chelating agents (such asethylenediamine tetraacetic acid (EDTA)), complexing agents (such ascaffeine, polyvinylpyrrolidone, beta-cyclodextrin, orhydroxypropyl-beta-cyclodextrin), fillers, monosaccharides,disaccharides, and other carbohydrates (such as glucose, mannose, ordextrins), proteins (such as serum albumin, gelatin, orimmunoglobulins), coloring, flavoring and diluting agents, emulsifyingagents, hydrophilic polymers (such as polyvinylpyrrolidone), lowmolecular weight polypeptides, salt-forming counterions (such assodium), preservatives (such as benzalkonium chloride, benzoic acid,salicylic acid, thimerosal, phenethyl alcohol, methylparaben,propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide),solvents (such as glycerin, propylene glycol, or polyethylene glycol),sugar alcohols (such as mannitol or sorbitol), suspending agents,surfactants or wetting agents (such as pluronics; PEG; sorbitan esters;polysorbates such as polysorbate 20 or polysorbate 80; triton;tromethamine; lecithin; cholesterol or tyloxapal), stability enhancingagents (such as sucrose or sorbitol), tonicity enhancing agents (such asalkali metal halides—preferably sodium or potassium chloride—or mannitolsorbitol), delivery vehicles, diluents, excipients and/or pharmaceuticaladjuvants. See Remington's Pharmaceutical Sciences (18th Ed., A. R.Gennaro, ed., Mack Publishing Company 1990.

The optimal pharmaceutical composition will be determined by a skilledartisan depending upon, for example, the intended route ofadministration, delivery format, and desired dosage. See, e.g.,Remington's Pharmaceutical Sciences, supra. Such compositions mayinfluence the physical state, stability, rate of in vivo release, andrate of in vivo clearance of the IFN-L molecule.

The primary vehicle or carrier in a pharmaceutical composition may beeither aqueous or non-aqueous in nature. For example, a suitable vehicleor carrier for injection may be water, physiological saline solution, orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. Neutralbuffered saline or saline mixed with serum albumin are further exemplaryvehicles. Other exemplary pharmaceutical compositions comprise Trisbuffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, whichmay further include sorbitol or a suitable substitute. In one embodimentof the present invention, IFN-L polypeptide compositions may be preparedfor storage by mixing the selected composition having the desired degreeof purity with optional formulation agents (Remington's PharmaceuticalSciences, supra) in the form of a lyophilized cake or an aqueoussolution. Further, the IFN-L polypeptide product may be formulated as alyophilizate using appropriate excipients such as sucrose.

The IFN-L polypeptide pharmaceutical compositions can be selected forparenteral delivery. Alternatively, the compositions may be selected forinhalation or for delivery through the digestive tract, such as orally.The preparation of such pharmaceutically acceptable compositions iswithin the skill of the art.

The formulation components are present in concentrations that areacceptable to the site of administration. For example, buffers are usedto maintain the composition at physiological pH or at a slightly lowerpH, typically within a pH range of from about 5 to about 8.

When parenteral administration is contemplated, the therapeuticcompositions for use in this invention may be in the form of apyrogen-free, parenterally acceptable, aqueous solution comprising thedesired IFN-L molecule in a pharmaceutically acceptable vehicle. Aparticularly suitable vehicle for parenteral injection is steriledistilled water in which an IFN-L molecule is formulated as a sterile,isotonic solution, properly preserved. Yet another preparation caninvolve the formulation of the desired molecule with an agent, such asinjectable microspheres, bio-erodible particles, polymeric compounds(such as polylactic acid or polyglycolic acid), beads, or liposomes,that provides for the controlled or sustained release of the productwhich may then be delivered via a depot injection. Hyaluronic acid mayalso be used, and this may have the effect of promoting sustainedduration in the circulation. Other suitable means for the introductionof the desired molecule include implantable drug delivery devices.

In one embodiment, a pharmaceutical composition may be formulated forinhalation. For example, IFN-L polypeptide may be formulated as a drypowder for inhalation. IFN-L polypeptide or nucleic acid moleculeinhalation solutions may also be formulated with a propellant foraerosol delivery. In yet another embodiment, solutions may be nebulized.Pulmonary administration is further described in PCT Pub. No. WO94/20069, which describes the pulmonary delivery of chemically modifiedproteins.

It is also contemplated that certain formulations may be administeredorally. In one embodiment of the present invention, IFN-L polypeptidesthat are administered in this fashion can be formulated with or withoutthose carriers customarily used in the compounding of solid dosage formssuch as tablets and capsules. For example, a capsule may be designed torelease the active portion of the formulation at the point in thegastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized. Additional agents can be includedto facilitate absorption of the IFN-L polypeptide. Diluents, flavorings,low melting point waxes, vegetable oils, lubricants, suspending agents,tablet disintegrating agents, and binders may also be employed.

Another pharmaceutical composition may involve an effective quantity ofIFN-L polypeptides in a mixture with non-toxic excipients that aresuitable for the manufacture of tablets. By dissolving the tablets insterile water, or another appropriate vehicle, solutions can be preparedin unit-dose form. Suitable excipients include, but are not limited to,inert diluents, such as calcium carbonate, sodium carbonate orbicarbonate, lactose, or calcium phosphate; or binding agents, such asstarch, gelatin, or acacia; or lubricating agents such as magnesiumstearate, stearic acid, or talc.

Additional IFN-L polypeptide pharmaceutical compositions will be evidentto those skilled in the art, including formulations involving IFN-Lpolypeptides in sustained- or controlled-delivery formulations.Techniques for formulating a variety of other sustained- orcontrolled-delivery means, such as liposome carriers, bio-erodiblemicroparticles or porous beads and depot injections, are also known tothose skilled in the art. See, e.g., PCT/US93/00829, which describes thecontrolled release of porous polymeric microparticles for the deliveryof pharmaceutical compositions.

Additional examples of sustained-release preparations includesemipermeable polymer matrices in the form of shaped articles, e.g.films, or microcapsules. Sustained release matrices may includepolyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 andEuropean Patent No. 058481), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al., 1983, Biopolymers 22:547-56),poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed.Mater. Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105),ethylene vinyl acetate (Langer et al., supra) orpoly-D(−)-3-hydroxybutyric acid (European Patent No. 133988).Sustained-release compositions may also include liposomes, which can beprepared by any of several methods known in the art. See, e.g., Eppsteinet al., 1985, Proc. Natl. Acad. Sci. USA 82:3688-92; and European PatentNos. 036676, 088046, and 143949.

The IFN-L pharmaceutical composition to be used for in vivoadministration typically must be sterile. This may be accomplished byfiltration through sterile filtration membranes. Where the compositionis lyophilized, sterilization using this method may be conducted eitherprior to, or following, lyophilization and reconstitution. Thecomposition for parenteral administration may be stored in lyophilizedform or in a solution. In addition, parenteral compositions generallyare placed into a container having a sterile access port, for example,an intravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

Once the pharmaceutical composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or as a dehydrated or lyophilized powder. Such formulations may bestored either in a ready-to-use form or in a form (e.g., lyophilized)requiring reconstitution prior to administration.

In a specific embodiment, the present invention is directed to kits forproducing a single-dose administration unit. The kits may each containboth a first container having a dried protein and a second containerhaving an aqueous formulation. Also included within the scope of thisinvention are kits containing single and multi-chambered pre-filledsyringes (e.g., liquid syringes and lyosyringes).

The effective amount of an IFN-L pharmaceutical composition to beemployed therapeutically will depend, for example, upon the therapeuticcontext and objectives. One skilled in the art will appreciate that theappropriate dosage levels for treatment will thus vary depending, inpart, upon the molecule delivered, the indication for which the IFN-Lmolecule is being used, the route of administration, and the size (bodyweight, body surface, or organ size) and condition (the age and generalhealth) of the patient. Accordingly, the clinician may titer the dosageand modify the route of administration to obtain the optimal therapeuticeffect. A typical dosage may range from about 0.1 μg/kg to up to about100 mg/kg or more, depending on the factors mentioned above. In otherembodiments, the dosage may range from 0.1 μg/kg up to about 100 mg/kg;or 1 μg/kg up to about 100 mg/kg; or 5 μg/kg up to about 100 mg/kg.

The frequency of dosing will depend upon the pharmacokinetic parametersof the IFN-L molecule in the formulation being used. Typically, aclinician will administer the composition until a dosage is reached thatachieves the desired effect. The composition may therefore beadministered as a single dose, as two or more doses (which may or maynot contain the same amount of the desired molecule) over time, or as acontinuous infusion via an implantation device or catheter. Furtherrefinement of the appropriate dosage is routinely made by those ofordinary skill in the art and is within the ambit of tasks routinelyperformed by them. Appropriate dosages may be ascertained through use ofappropriate dose-response data.

The route of administration of the pharmaceutical composition is inaccord with known methods, e.g., orally; through injection byintravenous, intraperitoneal, intracerebral (intraparenchymal),intracerebroventricular, intramuscular, intraocular, intraarterial,intraportal, or intralesional routes; by sustained release systems; orby implantation devices. Where desired, the compositions may beadministered by bolus injection or continuously by infusion, or byimplantation device.

Alternatively or additionally, the composition may be administeredlocally via implantation of a membrane, sponge, or other appropriatematerial onto which the desired molecule has been absorbed orencapsulated. Where an implantation device is used, the device may beimplanted into any suitable tissue or organ, and delivery of the desiredmolecule may be via diffusion, timed-release bolus, or continuousadministration.

In some cases, it may be desirable to use IFN-L polypeptidepharmaceutical compositions in an ex vivo manner. In such instances,cells, tissues, or organs that have been removed from the patient areexposed to IFN-L polypeptide pharmaceutical compositions after which thecells, tissues, or organs are subsequently implanted back into thepatient.

In other cases, an IFN-L polypeptide can be delivered by implantingcertain cells that have been genetically engineered, using methods suchas those described herein, to express and secrete the IFN-L polypeptide.Such cells may be animal or human cells, and may be autologous,heterologous, or xenogeneic. Optionally, the cells may be immortalized.In order to decrease the chance of an immunological response, the cellsmay be encapsulated to avoid infiltration of surrounding tissues. Theencapsulation materials are typically biocompatible, semi-permeablepolymeric enclosures or membranes that allow the release of the proteinproduct(s) but prevent the destruction of the cells by the patient'simmune system or by other detrimental factors from the surroundingtissues.

As discussed herein, it may be desirable to treat isolated cellpopulations (such as stem cells, lymphocytes, red blood cells,chondrocytes, neurons, and the like) with one or more IFN-Lpolypeptides. This can be accomplished by exposing the isolated cells tothe polypeptide directly, where it is in a form that is permeable to thecell membrane.

Additional embodiments of the present invention relate to cells andmethods (e.g., homologous recombination and/or other recombinantproduction methods) for both the in vitro production of therapeuticpolypeptides and for the production and delivery of therapeuticpolypeptides by gene therapy or cell therapy. Homologous and otherrecombination methods may be used to modify a cell that contains anormally transcriptionally-silent IFN-L gene, or an under-expressedgene, and thereby produce a cell which expresses therapeuticallyefficacious amounts of IFN-L polypeptides.

Homologous recombination is a technique originally developed fortargeting genes to induce or correct mutations in transcriptionallyactive genes. Kucherlapati, 1989, Prog. in Nucl. Acid Res. & Mol. Biol.36:301. The basic technique was developed as a method for introducingspecific mutations into specific regions of the mammalian genome (Thomaset al., 1986, Cell 44:419-28; Thomas and Capecchi, 1987, Cell 51:503-12;Doetschman et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:8583-87) or tocorrect specific mutations within defective genes (Doetschman et al.,1987, Nature 330:576-78). Exemplary homologous recombination techniquesare described in U.S. Pat. No. 5,272,071; European Patent Nos. 9193051and 505500; PCT/US90/07642, and PCT Pub No. WO 91/09955).

Through homologous recombination, the DNA sequence to be inserted intothe genome can be directed to a specific region of the gene of interestby attaching it to targeting DNA. The targeting DNA is a nucleotidesequence that is complementary (homologous) to a region of the genomicDNA. Small pieces of targeting DNA that are complementary to a specificregion of the genome are put in contact with the parental strand duringthe DNA replication process. It is a general property of DNA that hasbeen inserted into a cell to hybridize, and therefore, recombine withother pieces of endogenous DNA through shared homologous regions. Ifthis complementary strand is attached to an oligonucleotide thatcontains a mutation or a different sequence or an additional nucleotide,it too is incorporated into the newly synthesized strand as a result ofthe recombination. As a result of the proofreading function, it ispossible for the new sequence of DNA to serve as the template. Thus, thetransferred DNA is incorporated into the genome.

Attached to these pieces of targeting DNA are regions of DNA that mayinteract with or control the expression of an IFN-L polypeptide, e.g.,flanking sequences. For example, a promoter/enhancer element, asuppressor, or an exogenous transcription modulatory element is insertedin the genome of the intended host cell in proximity and orientationsufficient to influence the transcription of DNA encoding the desiredIFN-L polypeptide. The control element controls a portion of the DNApresent in the host cell genome. Thus, the expression of the desiredIFN-L polypeptide may be achieved not by transfection of DNA thatencodes the IFN-L gene itself, but rather by the use of targeting DNA(containing regions of homology with the endogenous gene of interest)coupled with DNA regulatory segments that provide the endogenous genesequence with recognizable signals for transcription of an IFN-L gene.

In an exemplary method, the expression of a desired targeted gene in acell (i.e., a desired endogenous cellular gene) is altered viahomologous recombination into the cellular genome at a preselected site,by the introduction of DNA which includes at least a regulatorysequence, an exon, and a splice donor site. These components areintroduced into the chromosomal (genomic) DNA in such a manner thatthis, in effect, results in the production of a new transcription unit(in which the regulatory sequence, the exon, and the splice donor sitepresent in the DNA construct are operatively linked to the endogenousgene). As a result of the introduction of these components into thechromosomal DNA, the expression of the desired endogenous gene isaltered.

Altered gene expression, as described herein, encompasses activating (orcausing to be expressed) a gene which is normally silent (unexpressed)in the cell as obtained, as well as increasing the expression of a genewhich is not expressed at physiologically significant levels in the cellas obtained. The embodiments further encompass changing the pattern ofregulation or induction such that it is different from the pattern ofregulation or induction that occurs in the cell as obtained, andreducing (including eliminating) the expression of a gene which isexpressed in the cell as obtained.

One method by which homologous recombination can be used to increase, orcause, IFN-L polypeptide production from a cell's endogenous IFN-L geneinvolves first using homologous recombination to place a recombinationsequence from a site-specific recombination system (e.g., Cre/loxP,FLP/FRT) (Sauer, 1994, Curr. Opin. Biotechnol., 5:521-27; Sauer, 1993,Methods Enzymol., 225:890-900) upstream of (i.e., 5′ to) the cell'sendogenous genomic IFN-L polypeptide coding region. A plasmid containinga recombination site homologous to the site that was placed justupstream of the genomic IFN-L polypeptide coding region is introducedinto the modified cell line along with the appropriate recombinaseenzyme. This recombinase causes the plasmid to integrate, via theplasmid's recombination site, into the recombination site located justupstream of the genomic IFN-L polypeptide coding region in the cell line(Baubonis and Sauer, 1993, Nucleic Acids Res. 21:2025-29; O'Gorman etal., 1991, Science 251:1351-55). Any flanking sequences known toincrease transcription (e.g., enhancer/promoter, intron, translationalenhancer), if properly positioned in this plasmid, would integrate insuch a manner as to create a new or modified transcriptional unitresulting in de novo or increased IFN-L polypeptide production from thecell's endogenous IFN-L gene.

A further method to use the cell line in which the site specificrecombination sequence had been placed just upstream of the cell'sendogenous genomic IFN-L polypeptide coding region is to use homologousrecombination to introduce a second recombination site elsewhere in thecell line's genome. The appropriate recombinase enzyme is thenintroduced into the two-recombination-site cell line, causing arecombination event (deletion, inversion, and translocation) (Sauer,1994, Curr. Opin. Biotechnol., 5:521-27; Sauer, 1993, Methods Enzymol.,225:890-900) that would create a new or modified transcriptional unitresulting in de novo or increased IFN-L polypeptide production from thecell's endogenous IFN-L gene.

An additional approach for increasing, or causing, the expression ofIFN-L polypeptide from a cell's endogenous IFN-L gene involvesincreasing, or causing, the expression of a gene or genes (e.g.,transcription factors) and/or decreasing the expression of a gene orgenes (e.g., transcriptional repressors) in a manner which results in denovo or increased IFN-L polypeptide production from the cell'sendogenous IFN-L gene. This method includes the introduction of anon-naturally occurring polypeptide (e.g., a polypeptide comprising asite specific DNA binding domain fused to a transcriptional factordomain) into the cell such that de novo or increased IFN-L polypeptideproduction from the cell's endogenous IFN-L gene results.

The present invention further relates to DNA constructs useful in themethod of altering expression of a target gene. In certain embodiments,the exemplary DNA constructs comprise: (a) one or more targetingsequences, (b) a regulatory sequence, (c) an exon, and (d) an unpairedsplice-donor site. The targeting sequence in the DNA construct directsthe integration of elements (a)-(d) into a target gene in a cell suchthat the elements (b)-(d) are operatively linked to sequences of theendogenous target gene. In another embodiment, the DNA constructscomprise: (a) one or more targeting sequences, (b) a regulatorysequence, (c) an exon, (d) a splice-donor site, (e) an intron, and (f) asplice-acceptor site, wherein the targeting sequence directs theintegration of elements (a)-(f) such that the elements of (b)-(f) areoperatively linked to the endogenous gene. The targeting sequence ishomologous to the preselected site in the cellular chromosomal DNA withwhich homologous recombination is to occur. In the construct, the exonis generally 3′ of the regulatory sequence and the splice-donor site is3′ of the exon.

If the sequence of a particular gene is known, such as the nucleic acidsequence of IFN-L polypeptide presented herein, a piece of DNA that iscomplementary to a selected region of the gene can be synthesized orotherwise obtained, such as by appropriate restriction of the native DNAat specific recognition sites bounding the region of interest. Thispiece serves as a targeting sequence upon insertion into the cell andwill hybridize to its homologous region within the genome. If thishybridization occurs during DNA replication, this piece of DNA, and anyadditional sequence attached thereto, will act as an Okazaki fragmentand will be incorporated into the newly synthesized daughter strand ofDNA. The present invention, therefore, includes nucleotides encoding anIFN-L polypeptide, which nucleotides may be used as targeting sequences.

IFN-L polypeptide cell therapy, e.g., the implantation of cellsproducing IFN-L polypeptides, is also contemplated. This embodimentinvolves implanting cells capable of synthesizing and secreting abiologically active form of IFN-L polypeptide. Such IFN-Lpolypeptide-producing cells can be cells that are natural producers ofIFN-L polypeptides or may be recombinant cells whose ability to produceIFN-L polypeptides has been augmented by transformation with a geneencoding the desired IFN-L polypeptide or with a gene augmenting theexpression of IFN-L polypeptide. Such a modification may be accomplishedby means of a vector suitable for delivering the gene as well aspromoting its expression and secretion. In order to minimize a potentialimmunological reaction in patients being administered an IFN-Lpolypeptide, as may occur with the administration of a polypeptide of aforeign species, it is preferred that the natural cells producing IFN-Lpolypeptide be of human origin and produce human IFN-L polypeptide.Likewise, it is preferred that the recombinant cells producing IFN-Lpolypeptide be transformed with an expression vector containing a geneencoding a human IFN-L polypeptide.

Implanted cells may be encapsulated to avoid the infiltration ofsurrounding tissue. Human or non-human animal cells may be implanted inpatients in biocompatible, semipermeable polymeric enclosures ormembranes that allow the release of IFN-L polypeptide, but that preventthe destruction of the cells by the patient's immune system or by otherdetrimental factors from the surrounding tissue. Alternatively, thepatient's own cells, transformed to produce IFN-L polypeptides ex vivo,may be implanted directly into the patient without such encapsulation.

Techniques for the encapsulation of living cells are known in the art,and the preparation of the encapsulated cells and their implantation inpatients may be routinely accomplished. For example, Baetge et al. (PCTPub. No. WO 95/05452 and PCT/US94/09299) describe membrane capsulescontaining genetically engineered cells for the effective delivery ofbiologically active molecules. The capsules are biocompatible and areeasily retrievable. The capsules encapsulate cells transfected withrecombinant DNA molecules comprising DNA sequences coding forbiologically active molecules operatively linked to promoters that arenot subject to down-regulation in vivo upon implantation into amammalian host. The devices provide for the delivery of the moleculesfrom living cells to specific sites within a recipient. In addition, seeU.S. Pat. Nos. 4,892,538; 5,011,472; and 5,106,627. A system forencapsulating living cells is described in PCT Pub. No. WO 91/10425(Aebischer et al.). See also, PCT Pub. No. WO 91/10470 (Aebischer etal.); Winn et al., 1991, Exper. Neurol. 113:322-29; Aebischer et al.,1991, Exper. Neurol. 111:269-75; and Tresco et al., 1992, ASAIO38:17-23.

In vivo and in vitro gene therapy delivery of IFN-L polypeptides is alsoenvisioned. One example of a gene therapy technique is to use the IFN-Lgene (either genomic DNA, cDNA, and/or synthetic DNA) encoding an IFN-Lpolypeptide which may be operably linked to a constitutive or induciblepromoter to form a “gene therapy DNA construct.” The promoter may behomologous or heterologous to the endogenous IFN-L gene, provided thatit is active in the cell or tissue type into which the construct will beinserted. Other components of the gene therapy DNA construct mayoptionally include DNA molecules designed for site-specific integration(e.g., endogenous sequences useful for homologous recombination),tissue-specific promoters, enhancers or silencers, DNA molecules capableof providing a selective advantage over the parent cell, DNA moleculesuseful as labels to identify transformed cells, negative selectionsystems, cell specific binding agents (as, for example, for celltargeting), cell-specific internalization factors, transcription factorsenhancing expression from a vector, and factors enabling vectorproduction.

A gene therapy DNA construct can then be introduced into cells (eitherex vivo or in vivo) using viral or non-viral vectors. One means forintroducing the gene therapy DNA construct is by means of viral vectorsas described herein. Certain vectors, such as retroviral vectors, willdeliver the DNA construct to the chromosomal DNA of the cells, and thegene can integrate into the chromosomal DNA. Other vectors will functionas episomes, and the gene therapy DNA construct will remain in thecytoplasm.

In yet other embodiments, regulatory elements can be included for thecontrolled expression of the IFN-L gene in the target cell. Suchelements are turned on in response to an appropriate effector. In thisway, a therapeutic polypeptide can be expressed when desired. Oneconventional control means involves the use of small molecule dimerizersor rapalogs to dimerize chimeric proteins which contain a smallmolecule-binding domain and a domain capable of initiating a biologicalprocess, such as a DNA-binding protein or transcriptional activationprotein (see PCT Pub. Nos. WO 96/41865, WO 97/31898, and WO 97/31899).The dimerization of the proteins can be used to initiate transcriptionof the transgene.

An alternative regulation technology uses a method of storing proteinsexpressed from the gene of interest inside the cell as an aggregate orcluster. The gene of interest is expressed as a fusion protein thatincludes a conditional aggregation domain that results in the retentionof the aggregated protein in the endoplasmic reticulum. The storedproteins are stable and inactive inside the cell. The proteins can bereleased, however, by administering a drug (e.g., small molecule ligand)that removes the conditional aggregation domain and thereby specificallybreaks apart the aggregates or clusters so that the proteins may besecreted from the cell. See Aridor et al., 2000, Science 287:816-17 andRivera et al., 2000, Science 287:826-30.

Other suitable control means or gene switches include, but are notlimited to, the systems described herein. Mifepristone (RU486) is usedas a progesterone antagonist. The binding of a modified progesteronereceptor ligand-binding domain to the progesterone antagonist activatestranscription by forming a dimer of two transcription factors that thenpass into the nucleus to bind DNA. The ligand-binding domain is modifiedto eliminate the ability of the receptor to bind to the natural ligand.The modified steroid hormone receptor system is further described inU.S. Pat. No. 5,364,791 and PCT Pub. Nos. WO 96/40911 and WO 97/10337.

Yet another control system uses ecdysone (a fruit fly steroid hormone)which binds to and activates an ecdysone receptor (cytoplasmicreceptor). The receptor then translocates to the nucleus to bind aspecific DNA response element (promoter from ecdysone-responsive gene).The ecdysone receptor includes a trans activation domain, DNA-bindingdomain, and ligand-binding domain to initiate transcription. Theecdysone system is further described in U.S. Pat. No. 5,514,578 and PCTPub. Nos. WO 97/38117, WO 96/37609, and WO 93/03162.

Another control means uses a positive tetracycline-controllabletransactivator. This system involves a mutated tet repressor proteinDNA-binding domain (mutated tet R-4 amino acid changes which resulted ina reverse tetracycline-regulated transactivator protein, i.e., it bindsto a tet operator in the presence of tetracycline) linked to apolypeptide which activates transcription. Such systems are described inU.S. Pat. Nos. 5,464,758, 5,650,298, and 5,654,168.

Additional expression control systems and nucleic acid constructs aredescribed in U.S. Pat. Nos. 5,741,679 and 5,834,186, to InnovirLaboratories Inc.

In vivo gene therapy may be accomplished by introducing the geneencoding IFN-L polypeptide into cells via local injection of an IFN-Lnucleic acid molecule or by other appropriate viral or non-viraldelivery vectors. Hefti, 1994, Neurobiology 25:1418-35. For example, anucleic acid molecule encoding an IFN-L polypeptide may be contained inan adeno-associated virus (AAV) vector for delivery to the targetedcells (see, e.g., Johnson, PCT Pub. No. WO 95/34670; PCT App. No.PCT/US95/07178). The recombinant AAV genome typically contains AAVinverted terminal repeats flanking a DNA sequence encoding an IFN-Lpolypeptide operably linked to functional promoter and polyadenylationsequences.

Alternative suitable viral vectors include, but are not limited to,retrovirus, adenovirus, herpes simplex virus, lentivirus, hepatitisvirus, parvovirus, papovavirus, poxvirus, alphavirus, coronavirus,rhabdovirus, paramyxovirus, and papilloma virus vectors. U.S. Pat. No.5,672,344 describes an in vivo viral-mediated gene transfer systeminvolving a recombinant neurotrophic HSV-1 vector. U.S. Pat. No.5,399,346 provides examples of a process for providing a patient with atherapeutic protein by the delivery of human cells which have beentreated in vitro to insert a DNA segment encoding a therapeutic protein.Additional methods and materials for the practice of gene therapytechniques are described in U.S. Pat. Nos. 5,631,236 (involvingadenoviral vectors), 5,672,510 (involving retroviral vectors), 5,635,399(involving retroviral vectors expressing cytokines).

Nonviral delivery methods include, but are not limited to,liposome-mediated transfer, naked DNA delivery (direct injection),receptor-mediated transfer (ligand-DNA complex), electroporation,calcium phosphate precipitation, and microparticle bombardment (e.g.,gene gun). Gene therapy materials and methods may also include induciblepromoters, tissue-specific enhancer-promoters, DNA sequences designedfor site-specific integration, DNA sequences capable of providing aselective advantage over the parent cell, labels to identify transformedcells, negative selection systems and expression control systems (safetymeasures), cell-specific binding agents (for cell targeting),cell-specific internalization factors, and transcription factors toenhance expression by a vector as well as methods of vector manufacture.Such additional methods and materials for the practice of gene therapytechniques are described in U.S. Pat. Nos. 4,970,154 (involvingelectroporation techniques), 5,679,559 (describing alipoprotein-containing system for gene delivery), 5,676,954 (involvingliposome carriers), 5,593,875 (describing methods for calcium phosphatetransfection), and 4,945,050 (describing a process wherein biologicallyactive particles are propelled at cells at a speed whereby the particlespenetrate the surface of the cells and become incorporated into theinterior of the cells), and PCT Pub. No. WO 96/40958 (involving nuclearligands).

It is also contemplated that IFN-L gene therapy or cell therapy canfurther include the delivery of one or more additional polypeptide(s) inthe same or a different cell(s). Such cells may be separately introducedinto the patient, or the cells may be contained in a single implantabledevice, such as the encapsulating membrane described above, or the cellsmay be separately modified by means of viral vectors.

A means to increase endogenous IFN-L polypeptide expression in a cellvia gene therapy is to insert one or more enhancer elements into theIFN-L polypeptide promoter, where the enhancer elements can serve toincrease transcriptional activity of the IFN-L gene. The enhancerelements used will be selected based on the tissue in which one desiresto activate the gene—enhancer elements known to confer promoteractivation in that tissue will be selected. For example, if a geneencoding an IFN-L polypeptide is to be “turned on” in T-cells, the lckpromoter enhancer element may be used. Here, the functional portion ofthe transcriptional element to be added may be inserted into a fragmentof DNA containing the IFN-L polypeptide promoter (and optionally,inserted into a vector and/or 5′ and/or 3′ flanking sequences) usingstandard cloning techniques. This construct, known as a “homologousrecombination construct,” can then be introduced into the desired cellseither ex vivo or in vivo.

Gene therapy also can be used to decrease IFN-L polypeptide expressionby modifying the nucleotide sequence of the endogenous promoter. Suchmodification is typically accomplished via homologous recombinationmethods. For example, a DNA molecule containing all or a portion of thepromoter of the IFN-L gene selected for inactivation can be engineeredto remove and/or replace pieces of the promoter that regulatetranscription. For example, the TATA box and/or the binding site of atranscriptional activator of the promoter may be deleted using standardmolecular biology techniques; such deletion can inhibit promoteractivity thereby repressing the transcription of the corresponding IFN-Lgene. The deletion of the TATA box or the transcription activatorbinding site in the promoter may be accomplished by generating a DNAconstruct comprising all or the relevant portion of the IFN-Lpolypeptide promoter (from the same or a related species as the IFN-Lgene to be regulated) in which one or more of the TATA box and/ortranscriptional activator binding site nucleotides are mutated viasubstitution, deletion and/or insertion of one or more nucleotides. As aresult, the TATA box and/or activator binding site has decreasedactivity or is rendered completely inactive. This construct, which alsowill typically contain at least about 500 bases of DNA that correspondto the native (endogenous) 5′ and 3′ DNA sequences adjacent to thepromoter segment that has been modified, may be introduced into theappropriate cells (either ex vivo or in vivo) either directly or via aviral vector as described herein. Typically, the integration of theconstruct into the genomic DNA of the cells will be via homologousrecombination, where the 5′ and 3′ DNA sequences in the promoterconstruct can serve to help integrate the modified promoter region viahybridization to the endogenous chromosomal DNA.

Therapeutic Uses

IFN-L nucleic acid molecules, polypeptides, and agonists and antagoniststhereof can be used to treat, diagnose, ameliorate, or prevent a numberof diseases, disorders, or conditions, including those recited herein.

IFN-L polypeptide agonists and antagonists include those molecules whichregulate IFN-L polypeptide activity and either increase or decrease atleast one activity of the mature form of the IFN-L polypeptide. Agonistsor antagonists may be co-factors, such as a protein, peptide,carbohydrate, lipid, or small molecular weight molecule, which interactwith IFN-L polypeptide and thereby regulate its activity. Potentialpolypeptide agonists or antagonists include antibodies that react witheither soluble or membrane-bound forms of IFN-L polypeptides thatcomprise part or all of the extracellular domains of the said proteins.Molecules that regulate IFN-L polypeptide expression typically includenucleic acids encoding IFN-L polypeptide that can act as anti-senseregulators of expression.

IFN-L polypeptides may play a role in controlling the growth andmaintenance of cancer cells based on the homology of IFN-L polypeptidesto known interferons. Accordingly, IFN-L nucleic acid molecules,polypeptides, and agonists and antagonists thereof may be useful for thediagnosis and/or treatment of cancer. Examples of such cancers include,but are not limited to, chronic myelogenous leukemia, hairy cellleukemia, Kaposi's sarcoma, melanomas, lung cancer, brain cancer, breastcancer, cancers of the hematopoetic system, prostate cancer, ovariancancer, and testicular cancer. Other cancers are encompassed within thescope of the invention

IFN-L polypeptides may play a role in the modulation of the immunesystem based on the homology of IFN-polypeptides to known interferons.Accordingly, IFN-L nucleic acid molecules, polypeptides, and agonistsand antagonists thereof may be useful for the diagnosis and/or treatmentof dysfunction of the immune system. Examples of such diseases include,but are not limited to, multiple sclerosis, rheumatoid arthritis,psoriatic arthritis, inflammatory arthritis, osteoarthritis,inflammatory joint disease, autoimmune disease, lupus, diabetes,inflammatory bowel disease, transplant rejection, and graft vs. hostdisease. Other diseases influenced by the dysfunction of the immunesystem are encompassed within the scope of the invention.

IFN-L polypeptides may play a role in the control of viral and microbialinfections based on the homology of IFN-polypeptides to knowninterferons. Accordingly, IFN-L nucleic acid molecules, polypeptides,and agonists and antagonists thereof may be useful for the diagnosisand/or treatment of infections. Examples of such diseases include, butare not limited to, hepatitis, human immunodeficiency virus, humanpapilloma virus, and chronic granulamatous. Other diseases caused byinfections are encompassed within the scope of the invention.

IFN-L polypeptides may play a role in the control of bone formation andmaintenance based on the homology of IFN-polypeptides to knowninterferons. Accordingly, IFN-L nucleic acid molecules, polypeptides,and agonists and antagonists may be useful for the diagnosis and/ortreatment of bone disorders. Examples of such diseases include, but arenot limited to, osteoporosis, osteopetrosis, osteogenesis imperfecta,Paget's disease, periodontal disease, and hypercalcemia. Other bonedisorders are encompassed within the scope of the invention.

IFN-L polypeptides may play a role in the inappropriate proliferation ofcells based on the homology of IFN-polypeptides to known interferons.Accordingly, IFN-L nucleic acid molecules, polypeptides, and agonistsand antagonists may be useful for the diagnosis and/or treatment ofdiseases where there is abnormal cell proliferation. Examples of suchdiseases include, but are not limited to, arteriosclerosis and vascularrestenosis. Other diseases influenced by the inappropriate proliferationof cells are encompassed within the scope of the invention.

In a specific embodiment, the present invention is directed to the useof an IFN-L polypeptide in combination (pretreatment, post-treatment, orconcurrent treatment) with secreted or soluble human fas antigen orrecombinant versions thereof (PCT Pub. No. WO 96/20206; Mountz et al.,1995, J. Immunol., 155:4829-37; and European Patent No. 510691). PCTPub. No. WO 96/20206 discloses secreted human fas antigen (native andrecombinant, including an Ig fusion protein), methods for isolating thegenes responsible for coding the soluble recombinant human fas antigen,methods for cloning the gene in suitable vectors and cell types, andmethods for expressing the gene to produce the inhibitors. EuropeanPatent No. 510691 teaches nucleic acids coding for human fas antigen,including soluble fas antigen, vectors expressing for said nucleicacids, and transformants transfected with the vector. When administeredparenterally, doses of a secreted or soluble fas antigen fusion proteineach are generally from about 1 μg/kg to about 100 μg/kg.

Treatment of the diseases and disorders recited herein can include theuse of first line drugs for control of pain and inflammation; thesedrugs are classified as non-steroidal, anti-inflammatory drugs (NSAIDs).Secondary treatments include corticosteroids, slow acting antirheumaticdrugs (SAARDs), or disease modifying (DM) drugs. Information regardingthe following compounds can be found in The Merck Manual of Diagnosisand Therapy (16th ed. 1992) and in Pharmaprojects (PJB PublicationsLtd).

In a specific embodiment, the present invention is directed to the useof an IFN-L polypeptide and any of one or more NSAIDs for the treatmentof the diseases and disorders recited herein, including acute andchronic inflammation such as rheumatic diseases, and graft versus hostdisease. NSAIDs owe their anti-inflammatory action, at least in part, tothe inhibition of prostaglandin synthesis (Goodman and Gilman, ThePharmacological Basis of Therapeutics (7th ed. 1985)). NSAIDs can becharacterized into at least nine groups: (1) salicylic acid derivatives,(2) propionic acid derivatives, (3) acetic acid derivatives, (4) fenamicacid derivatives, (5) carboxylic acid derivatives, (6) butyric acidderivatives, (7) oxicams, (8) pyrazoles, and (9) pyrazolones.

In another specific embodiment, the present invention is directed to theuse of an IFN-L polypeptide in combination (pretreatment,post-treatment, or concurrent treatment) with any of one or moresalicylic acid derivatives, prodrug esters, or pharmaceuticallyacceptable salts thereof. Such salicylic acid derivatives, prodrugesters, and pharmaceutically acceptable salts thereof comprise:acetaminosalol, aloxiprin, aspirin, benorylate, bromosaligenin, calciumacetylsalicylate, choline magnesium trisalicylate, magnesium salicylate,choline salicylate, diflusinal, etersalate, fendosal, gentisic acid,glycol salicylate, imidazole salicylate, lysine acetylsalicylate,mesalamine, morpholine salicylate, 1-naphthyl salicylate, olsalazine,parsalmide, phenyl acetylsalicylate, phenyl salicylate, salacetamide,salicylamide O-acetic acid, salsalate, sodium salicylate andsulfasalazine. Structurally related salicylic acid derivatives havingsimilar analgesic and anti-inflammatory properties are also intended tobe encompassed by this group.

In an additional specific embodiment, the present invention is directedto the use of an IFN-L polypeptide in combination (pretreatment,post-treatment, or concurrent treatment) with any of one or morepropionic acid derivatives, prodrug esters, or pharmaceuticallyacceptable salts thereof. The propionic acid derivatives, prodrugesters, and pharmaceutically acceptable salts thereof comprise:alminoprofen, benoxaprofen, bucloxic acid, carprofen, dexindoprofen,fenoprofen, flunoxaprofen, fluprofen, flurbiprofen, furcloprofen,ibuprofen, ibuprofen aluminum, ibuproxam, indoprofen, isoprofen,ketoprofen, loxoprofen, miroprofen, naproxen, naproxen sodium,oxaprozin, piketoprofen, pimeprofen, pirprofen, pranoprofen, protizinicacid, pyridoxiprofen, suprofen, tiaprofenic acid and tioxaprofen.Structurally related propionic acid derivatives having similar analgesicand anti-inflammatory properties are also intended to be encompassed bythis group.

In yet another specific embodiment, the present invention is directed tothe use of an IFN-L polypeptide in combination (pretreatment,post-treatment, or concurrent treatment) with any of one or more aceticacid derivatives, prodrug esters, or pharmaceutically acceptable saltsthereof. The acetic acid derivatives, prodrug esters, andpharmaceutically acceptable salts thereof comprise: acemetacin,alclofenac, amfenac, bufexamac, cinmetacin, clopirac, delmetacin,diclofenac potassium, diclofenac sodium, etodolac, felbinac,fenclofenac, fenclorac, fenclozic acid, fentiazac, furofenac,glucametacin, ibufenac, indomethacin, isofezolac, isoxepac, lonazolac,metiazinic acid, oxametacin, oxpinac, pimetacin, proglumetacin,sulindac, talmetacin, tiaramide, tiopinac, tolmetin, tolmetin sodium,zidometacin and zomepirac. Structurally related acetic acid derivativeshaving similar analgesic and anti-inflammatory properties are alsointended to be encompassed by this group.

In another specific embodiment, the present invention is directed to theuse of an IFN-L polypeptide in combination (pretreatment,post-treatment, or concurrent treatment) with any of one or more fenamicacid derivatives, prodrug esters, or pharmaceutically acceptable saltsthereof. The fenamic acid derivatives, prodrug esters, andpharmaceutically acceptable salts thereof comprise: enfenamic acid,etofenamate, flufenamic acid, isonixin, meclofenamic acid, meclofenamatesodium, medofenamic acid, mefenamic acid, niflumic acid, talniflumate,terofenamate, tolfenamic acid and ufenamate. Structurally relatedfenamic acid derivatives having similar analgesic and anti-inflammatoryproperties are also intended to be encompassed by this group.

In an additional specific embodiment, the present invention is directedto the use of an IFN-L polypeptide in combination (pretreatment,post-treatment, or concurrent treatment) with any of one or morecarboxylic acid derivatives, prodrug esters, or pharmaceuticallyacceptable salts thereof. The carboxylic acid derivatives, prodrugesters, and pharmaceutically acceptable salts thereof which can be usedcomprise: clidanac, diflunisal, flufenisal, inoridine, ketorolac andtinoridine. Structurally related carboxylic acid derivatives havingsimilar analgesic and anti-inflammatory properties are also intended tobe encompassed by this group.

In yet another specific embodiment, the present invention is directed tothe use of an IFN-L polypeptide in combination (pretreatment,post-treatment, or concurrent treatment) with any of one or more butyricacid derivatives, prodrug esters, or pharmaceutically acceptable saltsthereof. The butyric acid derivatives, prodrug esters, andpharmaceutically acceptable salts thereof comprise: bumadizon,butibufen, fenbufen and xenbucin. Structurally related butyric acidderivatives having similar analgesic and anti-inflammatory propertiesare also intended to be encompassed by this group.

In another specific embodiment, the present invention is directed to theuse of an IFN-L polypeptide in combination (pretreatment,post-treatment, or concurrent treatment) with any of one or moreoxicams, prodrug esters, or pharmaceutically acceptable salts thereof.The oxicams, prodrug esters, and pharmaceutically acceptable saltsthereof comprise: droxicam, enolicam, isoxicam, piroxicam, sudoxicam,tenoxicam and 4-hydroxyl-1,2-benzothiazine 1,1-dioxide4-(N-phenyl)-carboxamide. Structurally related oxicams having similaranalgesic and anti-inflammatory properties are also intended to beencompassed by this group.

In still another specific embodiment, the present invention is directedto the use of an IFN-L polypeptide in combination (pretreatment,post-treatment, or concurrent treatment) with any of one or morepyrazoles, prodrug esters, or pharmaceutically acceptable salts thereof.The pyrazoles, prodrug esters, and pharmaceutically acceptable saltsthereof which may be used comprise: difenamizole and epirizole.Structurally related pyrazoles having similar analgesic andanti-inflammatory properties are also intended to be encompassed by thisgroup.

In an additional specific embodiment, the present invention is directedto the use of an IFN-L polypeptide in combination (pretreatment,post-treatment or, concurrent treatment) with any of one or morepyrazolones, prodrug esters, or pharmaceutically acceptable saltsthereof. The pyrazolones, prodrug esters, and pharmaceuticallyacceptable salts thereof which may be used comprise: apazone,azapropazone, benzpiperylon, feprazone, mofebutazone, morazone,oxyphenbutazone, phenylbutazone, pipebuzone, propylphenazone,ramifenazone, suxibuzone and thiazolinobutazone. Structurally relatedpyrazalones having similar analgesic and anti-inflammatory propertiesare also intended to be encompassed by this group.

In another specific embodiment, the present invention is directed to theuse of an IFN-L polypeptide in combination (pretreatment,post-treatment, or concurrent treatment) with any of one or more of thefollowing: NSAIDs: ε-acetamidocaproic acid, S-adenosylmethionine,3-amino-4-hydroxybutyric acid, amixetrine, anitrazafen, antrafenine,bendazac, bendazac lysinate, benzydamine, beprozin, broperamole,bucolome, bufezolac, ciproquazone, cloximate, dazidamine, deboxamet,detomidine, difenpiramide, difenpyramide, difisalamine, ditazol,emorfazone, fanetizole mesylate, fenflumizole, floctafenine, flumizole,flunixin, fluproquazone, fopirtoline, fosfosal, guaimesal, guaiazolene,isonixirn, lefetamine HCl, leflunomide, lofemizole, lotifazole, lysinclonixinate, meseclazone, nabumetone, nictindole, nimesulide, orgotein,orpanoxin, oxaceprol, oxapadol, paranyline, perisoxal, perisoxalcitrate, pifoxime, piproxen, pirazolac, pirfenidone, proquazone,proxazole, thielavin B, tiflamizole, timegadine, tolectin, tolpadol,tryptamid and those designated by company code number such as 480156S,AA861, AD1590, AFP802, AFP860, AI77B, AP504, AU8001, BPPC, BW540C,CHINOIN 127, CN100, EB382, EL508, F1044, FK-506, GV3658, ITF182,KCNTEI6090, KME4, LA2851, MR714, MR897, MY309, ONO3144, PR823, PV102,PV108, R830, RS2131, SCR152, SH440, SIR133, SPAS510, SQ27239, ST281,SY6001, TA60, TAI-901 (4-benzoyl-1-indancarboxylic acid), TVX2706,U60257, UR2301 and WY41770. Structurally related NSAIDs having similaranalgesic and anti-inflammatory properties to the NSAIDs are alsointended to be encompassed by this group.

In still another specific embodiment, the present invention is directedto the use of an IFN-L polypeptide in combination (pretreatment,post-treatment or concurrent treatment) with any of one or morecorticosteroids, prodrug esters, or pharmaceutically acceptable saltsthereof for the treatment of the diseases and disorders recited herein,including acute and chronic inflammation such as rheumatic diseases,graft versus host disease, and multiple sclerosis. Corticosteroids,prodrug esters, and pharmaceutically acceptable salts thereof includehydrocortisone and compounds which are derived from hydrocortisone, suchas 21-acetoxypregnenolone, alclometasone, algestone, amcinonide,beclomethasone, betamethasone, betamethasone valerate, budesonide,chloroprednisone, clobetasol, clobetasol propionate, clobetasone,clobetasone butyrate, clocortolone, cloprednol, corticosterone,cortisone, cortivazol, deflazacon, desonide, desoximerasone,dexamethasone, diflorasone, diflucortolone, difluprednate, enoxolone,fluazacort, flucloronide, flumethasone, flumethasone pivalate,flucinolone acetonide, flunisolide, fluocinonide, fluorocinoloneacetonide, fluocortin butyl, fluocortolone, fluocortolone hexanoate,diflucortolone valerate, fluorometholone, fluperolone acetate,fluprednidene acetate, fluprednisolone, flurandenolide, formocortal,halcinonide, halometasone, halopredone acetate, hydrocortamate,hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate,hydrocortisone phosphate, hydrocortisone 21-sodium succinate,hydrocortisone tebutate, mazipredone, medrysone, meprednisone,methylprednisolone, mometasone furoate, paramethasone, prednicarbate,prednisolone, prednisolone 21-diedryaminoacetate, prednisolone sodiumphosphate, prednisolone sodium succinate, prednisolone sodium21-m-sulfobenzoate, prednisolone sodium 21-stearoglycolate, prednisolonetebutate, prednisolone 21-trimethylacetate, prednisone, prednival,prednylidene, prednylidene 21-diethylaminoacetate, tixocortol,triamcinolone, triamcinolone acetonide, triamcinolone benetonide andtriamcinolone hexacetonide. Structurally related corticosteroids havingsimilar analgesic and anti-inflammatory properties are also intended tobe encompassed by this group.

In another specific embodiment, the present invention is directed to theuse of an IFN-L polypeptide in combination (pretreatment,post-treatment, or concurrent treatment) with any of one or moreslow-acting antirheumatic drugs (SAARDs) or disease modifyingantirheumatic drugs (DMARDS), prodrug esters, or pharmaceuticallyacceptable salts thereof for the treatment of the diseases and disordersrecited herein, including acute and chronic inflammation such asrheumatic diseases, graft versus host disease, and multiple sclerosis.SAARDs or DMARDS, prodrug esters, and pharmaceutically acceptable saltsthereof comprise: allocupreide sodium, auranofin, aurothioglucose,aurothioglycanide, azathioprine, brequinar sodium, bucillamine, calcium3-aurothio-2-propanol-1-sulfonate, chlorambucil, chloroquine,clobuzarit, cuproxoline, cyclophosphamide, cyclosporin, dapsone,15-deoxyspergualin, diacerein, glucosamine, gold salts (e.g., cycloquinegold salt, gold sodium thiomalate, gold sodium thiosulfate),hydroxychloroquine, hydroxychloroquine sulfate, hydroxyurea, kebuzone,levamisole, lobenzarit, melittin, 6-mercaptopurine, methotrexate,mizoribine, mycophenolate mofetil, myoral, nitrogen mustard,D-penicillamine, pyridinol imidazoles such as SKNF86002 and SB203580,rapamycin, thiols, thymopoietin and vincristine. Structurally relatedSAARDs or DMARDs having similar analgesic and anti-inflammatoryproperties are also intended to be encompassed by this group.

In another specific embodiment, the present invention is directed to theuse of an IFN-L polypeptide in combination (pretreatment,post-treatment, or concurrent treatment) with any of one or more COX2inhibitors, prodrug esters, or pharmaceutically acceptable salts thereoffor the treatment of the diseases and disorders recited herein,including acute and chronic inflammation. Examples of COX2 inhibitors,prodrug esters, or pharmaceutically acceptable salts thereof include,for example, celecoxib. Structurally related COX2 inhibitors havingsimilar analgesic and anti-inflammatory properties are also intended tobe encompassed by this group.

In still another specific embodiment, the present invention is directedto the use of an IFN-L polypeptide in combination (pretreatment,post-treatment, or concurrent treatment) with any of one or moreantimicrobials, prodrug esters, or pharmaceutically acceptable saltsthereof for the treatment of the diseases and disorders recited herein,including acute and chronic inflammation. Antimicrobials include, forexample, the broad classes of penicillins, cephalosporins and otherbeta-lactams, aminoglycosides, azoles, quinolones, macrolides,rifamycins, tetracyclines, sulfonamides, lincosamides and polymyxins.The penicillins include, but are not limited to, penicillin G,penicillin V, methicillin, nafcillin, oxacillin, cloxacillin,dicloxacillin, floxacillin, ampicillin, ampicillin/sulbactam,amoxicillin, amoxicillin/clavulanate, hetacillin, cyclacillin,bacampicillin, carbenicillin, carbenicillin indanyl, ticarcillin,ticarcillin/clavulanate, azlocillin, mezlocillin, peperacillin, andmecillinam. The cephalosporins and other beta-lactams include, but arenot limited to, cephalothin, cephapirin, cephalexin, cephradine,cefazolin, cefadroxil, cefaclor, cefamandole, cefotetan, cefoxitin,ceruroxime, cefonicid, ceforadine, cefixime, cefotaxime, moxalactam,ceftizoxime, cetriaxone, cephoperazone, ceftazidime, imipenem andaztreonam. The aminoglycosides include, but are not limited to,streptomycin, gentamicin, tobramycin, amikacin, netilmicin, kanamycinand neomycin. The azoles include, but are not limited to, fluconazole.The quinolones include, but are not limited to, nalidixic acid,norfloxacin, enoxacin, ciprofloxacin, ofloxacin, sparfloxacin andtemafloxacin. The macrolides include, but are not limited to,erythomycin, spiramycin and azithromycin. The rifamycins include, butare not limited to, rifampin. The tetracyclines include, but are notlimited to, spicycline, chlortetracycline, clomocycline, demeclocycline,deoxycycline, guamecycline, lymecycline, meclocycline, methacycline,minocycline, oxytetracycline, penimepicycline, pipacycline,rolitetracycline, sancycline, senociclin and tetracycline. Thesulfonamides include, but are not limited to, sulfanilamide,sulfamethoxazole, sulfacetamide, sulfadiazine, sulfisoxazole andco-trimoxazole (trimethoprim/sulfamethoxazole). The lincosamidesinclude, but are not limited to, clindamycin and lincomycin. Thepolymyxins (polypeptides) include, but are not limited to, polymyxin Band colistin.

Agonists or antagonists of IFN-L polypeptide function may be used(simultaneously or sequentially) in combination with one or morecytokines, growth factors, antibiotics, anti-inflammatories, and/orchemotherapeutic agents as is appropriate for the condition beingtreated.

Other diseases caused by or mediated by undesirable levels of IFN-Lpolypeptides are encompassed within the scope of the invention.Undesirable levels include excessive levels of IFN-L polypeptides andsub-normal levels of IFN-L polypeptides.

Uses of IFN-L Nucleic Acids and Polypeptides

Nucleic acid molecules of the invention (including those that do notthemselves encode biologically active polypeptides) may be used to mapthe locations of the IFN-L gene and related genes on chromosomes.Mapping may be done by techniques known in the art, such as PCRamplification and in situ hybridization.

IFN-L nucleic acid molecules (including those that do not themselvesencode biologically active polypeptides), may be useful as hybridizationprobes in diagnostic assays to test, either qualitatively orquantitatively, for the presence of an IFN-L nucleic acid molecule inmammalian tissue or bodily fluid samples.

Other methods may also be employed where it is desirable to inhibit theactivity of one or more IFN-L polypeptides. Such inhibition may beeffected by nucleic acid molecules that are complementary to andhybridize to expression control sequences (triple helix formation) or toIFN-L mRNA. For example, antisense DNA or RNA molecules, which have asequence that is complementary to at least a portion of an IFN-L genecan be introduced into the cell. Anti-sense probes may be designed byavailable techniques using the sequence of the IFN-L gene disclosedherein. Typically, each such antisense molecule will be complementary tothe start site (5′ end) of each selected IFN-L gene. When the antisensemolecule then hybridizes to the corresponding IFN-L mRNA, translation ofthis mRNA is prevented or reduced. Anti-sense inhibitors provideinformation relating to the decrease or absence of an IFN-L polypeptidein a cell or organism.

Alternatively, gene therapy may be employed to create adominant-negative inhibitor of one or more IFN-L polypeptides. In thissituation, the DNA encoding a mutant polypeptide of each selected IFN-Lpolypeptide can be prepared and introduced into the cells of a patientusing either viral or non-viral methods as described herein. Each suchmutant is typically designed to compete with endogenous polypeptide inits biological role.

In addition, an IFN-L polypeptide, whether biologically active or not,may be used as an immunogen, that is, the polypeptide contains at leastone epitope to which antibodies may be raised. Selective binding agentsthat bind to an IFN-L polypeptide (as described herein) may be used forin vivo and in vitro diagnostic purposes, including, but not limited to,use in labeled form to detect the presence of IFN-L polypeptide in abody fluid or cell sample. The antibodies may also be used to prevent,treat, or diagnose a number of diseases and disorders, including thoserecited herein. The antibodies may bind to an IFN-L polypeptide so as todiminish or block at least one activity characteristic of an IFN-Lpolypeptide, or may bind to a polypeptide to increase at least oneactivity characteristic of an IFN-L polypeptide (including by increasingthe pharmacokinetics of the IFN-L polypeptide).

The IFN-L polypeptides of the present invention can be used to cloneIFN-L polypeptide receptors, using an expression cloning strategy.Radiolabeled (¹²⁵Iodine) IFN-L polypeptide or affinity/activity-taggedIFN-L polypeptide (such as an Fc fusion or an alkaline phosphatasefusion) can be used in binding assays to identify a cell type or cellline or tissue that expresses IFN-L polypeptide receptors. RNA isolatedfrom such cells or tissues can be converted to cDNA, cloned into amammalian expression vector, and transfected into mammalian cells (suchas COS or 293 cells) to create an expression library. A radiolabeled ortagged IFN-L polypeptide can then be used as an affinity ligand toidentify and isolate from this library the subset of cells that expressthe IFN-L polypeptide receptors on their surface. DNA can then beisolated from these cells and transfected into mammalian cells to createa secondary expression library in which the fraction of cells expressingIFN-L polypeptide receptors is many-fold higher than in the originallibrary. This enrichment process can be repeated iteratively until asingle recombinant clone containing an IFN-L polypeptide receptor isisolated. Isolation of the IFN-L polypeptide receptors is useful foridentifying or developing novel agonists and antagonists of the IFN-Lpolypeptide signaling pathway. Such agonists and antagonists includesoluble IFN-L polypeptide receptors, anti-IFN-L polypeptide receptorantibodies, small molecules, or antisense oligonucleotides, and they maybe used for treating, preventing, or diagnosing one or more of thediseases or disorders described herein.

A deposit of cDNA encoding human IFN-L polypeptide, subcloned intopSPORT1 (Gibco BRL) and transfected into E. coli strain DH10B, havingAccession No. PTA-976, were made with the American Type CultureCollection, 10801 University Boulevard, Manassas, Va. 20110-2209 on Nov.23, 1999.

The following examples are intended for illustration purposes only, andshould not be construed as limiting the scope of the invention in anyway.

Example 1 Cloning of the Rat IFN-L Polypeptide Gene

Generally, materials and methods as described in Sambrook et al. suprawere used to clone and analyze the gene encoding rat IFN-L polypeptide.

Sequences encoding the rat IFN-L polypeptide were isolated from a ratplacenta cDNA library by large scale random cDNA sequencing incombination with computer-assisted analysis. To construct the ratplacenta cDNA library, rat embryo day 17 [E17] placenta mRNA wasprepared by standard methods (Chomczynski and Sacchi, 1987, Anal.Biochem. 162:156). Following synthesis using the Superscript PlasmidcDNA kit (Gibco BRL), rat cDNA was subcloned into the Sal I and Not Isites of the pSPORT1 vector (Gibco BRL).

Sequence analysis of the full-length cDNA for rat IFN-L polypeptideindicated that the gene comprises a 573 bp open reading frame encoding aprotein of 191 amino acids (FIGS. 1A-1B). The rat IFN-L polypeptidesequence is predicted to contain a signal peptide (FIG. 1A, predictedsignal peptide indicated by underline). The rat IFN-L polypeptidesequence was identified as being a novel member of the interferon familyof proteins following comparisons of the rat IFN-L polypeptide sequencewith protein sequences in the GenBank database.

Example 2 Cloning of the Human IFN-L Polypeptide Gene

Generally, materials and methods as described in Sambrook et al. suprawere used to clone and analyze the gene encoding human IFN-Lpolypeptide.

An examination of the genomic structure of known members of theInterferon gene family revealed that members of this family share aunique intronless structure. Sequences encoding the human IFN-Lpolypeptide were, therefore, isolated by screening a human genomic DNAlibrary with a probe derived from the rat IFN-L polypeptide gene.

A radioactive rat IFN-L probe was generated by polymerase chain reaction(PCR) amplification of rat IFN-L polypeptide cDNA. Polymerase chainreactions (PCR) were performed using a Perkin-Elmer 9600 thermocycler(PE Biosystems, Foster City, Calif.) and the following reactionconditions: 20 ng of rat IFN-L polypeptide cDNA, 20 pmol each of primers1795-01 (5′-A-T-G-A-C-A-C-T-G-A-A-G-T-A-T-T-T-A-T-G-G-3′; SEQ ID NO: 20)and 1795-02 (5′-A-T-T-C-A-T-G-T-T-G-A-G-T-A-G-T-T-T-G-T-A-3′; SEQ ID NO:21), 1 mmol each of dATP, dTTP, dGTP, 0.01 mmol dCTP, 100 μCi ³²P-dCTP,4 mM MgCl₂, 1×PCR buffer, and SU Taq polymerase (PE Biosystems). A“cold” PCR reaction (i.e., one not performed in the presence ofradioactively labeled dCTP, and utilizing a balanced dNTP mix) wasprepared simultaneously with the labeled reaction. Amplificationreactions were carried out at 94° C. for 30 seconds, 60° C. for 30seconds, and 72° C. for 1 minute for 45 cycles. Pooled labeled andunlabeled probe was purified using a Quick Spin G-50 column (Qiagen),boiled at 100° C. for 10 minutes, and chilled on ice for 20 minutesprior to addition to the hybridization solution. Probes with a specificactivity of at least 5×10⁵ cpm/μL were generated using this method.

Sequences encoding the human IFN-L polypeptide were isolated byscreening a human lambda genomic DNA library (Stratagene, Cat. No.946206). For the primary screen, 1×10⁶ clones were plated at a densityof 50,000 colonies/plate and transferred to nitrocellulose filters usingstandard techniques. Positive clones were re-screened prior to analysis.

The rat IFN-L probe was hybridized to the filters overnight at 42° C. in30% formamide, 5×SSC, 2× Denhart's, 10 μg/mL salmon sperm DNA, 0.2% SDS,2 mM EDTA, and 0.1% pyrophosphate. Following hybridization, filters werewashed for 30-60 minutes at room temperature in 1×SSC and 0.1% SDS andthen for 15 minutes at 55° C. in 0.2×SSC and 0.1% SDS.

Three positive clones were recovered following primary and secondaryscreening, and lambda phage DNA was prepared by a solid plate culturemethod. The Not I insert was excised from the clones and ligated intopSPORT1 (Gibco BRL), and these ligations were subsequently used totransform E. coli strain DH10. Following transformation, plasmids wererecovered using a Spin Column plasmid prep kit (Qiagen).

Plasmids derived from the three positive genomic DNA clones wereanalyzed by Southern blot analysis using the rat IFN-L probe utilized inthe genomic DNA library screening. After digesting the recovered plasmidDNA with Hind III, the digested fragments were resolved on an agarosegel, and then transferred to a nylon membrane. Hybridization conditionswere identical to those utilized in the genomic DNA library screen.Southern blot analysis indicated that the three positive genomic cloneswere likely to contain identical genomic inserts. The fragmentshybridizing with the rat IFN-L probe were subsequently subcloned intopSPORT1 for sequencing analysis. This analysis confirmed that the threepositive genomic DNA clones contained identical genomic inserts.

Sequence analysis of the three genomic clones containing sequencesencoding human IFN-L polypeptide indicated that the gene comprises a 621bp open reading frame encoding a protein of 207 amino acids (FIGS.2A-2B). The human IFN-L polypeptide sequence is predicted to contain asignal peptide (FIG. 2A, predicted signal peptide indicated byunderline). Sequence analysis of IFN-L polypeptide strongly suggeststhat the protein is a secreted cytokine molecule.

A similarity of 64% was observed between the open reading frame of thehuman IFN-L gene and that of the rat IFN-L cDNA. FIG. 3 illustrates theamino acid sequence alignment of human IFN-L polypeptide (SEQ ID NO: 2),human IFN-β (SEQ ID NO: 7), and rat IFN-L polypeptide (SEQ ID NO: 4).Human IFN-L polypeptide is 30% identical to human IFN-β. Human IFN-Lpolypeptide is 40.5% identical to and 50% similar to rat IFN-Lpolypeptide. All five predicted cysteine residues in human IFN-Lpolypeptide are perfectly aligned with those in rat IFN-L polypeptide.

Example 3 IFN-L mRNA Expression

Developmental expression patterns of IFN-L mRNA were determined byNorthern blot analysis using a ³²P-labeled full-length rat cDNA probe todetect the presence of the IFN-L polypeptide transcript in severaldifferent stages of mouse and rat embryos. RNA was isolated from the ratand mouse embryos using the same techniques employed for theconstruction of the rat placenta cDNA library. Northern blots wereprehybridized in 40% formamide, 5×SSC, 1 mM EDTA, and 0.1% for 4 hoursat 42° C. The blots were hybridized overnight at 42° C. in the samesolution, except for the addition of the rat IFN-L probe. Followinghybridization, blots were washed for 30 minutes at 60° C. in 1×SSC and0.1% SDS.

Expression of IFN-L mRNA was examined in various human tissues by RT-PCRusing standard techniques. Human IFN-L mRNA was detected in pancreas,small intestine, prostrate, uterus, thyroid, and placenta.

The expression of IFN-L mRNA is localized by in situ hybridization. Apanel of normal embryonic and adult mouse tissues is fixed in 4%paraformaldehyde, embedded in paraffin, and sectioned at 5 μm. Sectionedtissues are permeabilized in 0.2 M HCl, digested with Proteinase K, andacetylated with triethanolamine and acetic anhydride. Sections areprehybridized for 1 hour at 60° C. in hybridization solution (300 mMNaCl, 20 mM Tris-HCl, pH 8.0, 5 mM EDTA, 1×Denhardt's solution, 0.2%SDS, 10 mM DTT, 0.25 mg/ml tRNA, 25 μg/ml polyA, 25 μg/ml polyC and 50%formamide) and then hybridized overnight at 60° C. in the same solutioncontaining 10% dextran and 2×10⁴ cpm/μl of a ³³P-labeled antisenseriboprobe complementary to the human IFN-L gene. The riboprobe isobtained by in vitro transcription of a clone containing human IFN-LcDNA sequences using standard techniques.

Following hybridization, sections are rinsed in hybridization solution,treated with RNaseA to digest unhybridized probe, and then washed in0.1×SSC at 55° C. for 30 minutes. Sections are then immersed in NTB-2emulsion (Kodak, Rochester, N.Y.), exposed for 3 weeks at 4° C.,developed, and counterstained with hematoxylin and eosin. Tissuemorphology and hybridization signal are simultaneously analyzed bydarkfield and standard illumination for brain (one sagittal and twocoronal sections), gastrointestinal tract (esophagus, stomach, duodenum,jejunum, ileum, proximal colon, and distal colon), pituitary, liver,lung, heart, spleen, thymus, lymph nodes, kidney, adrenal, bladder,pancreas, salivary gland, male and female reproductive organs (ovary,oviduct, and uterus in the female; and testis, epididymus, prostate,seminal vesicle, and vas deferens in the male), BAT and WAT(subcutaneous, peri-renal), bone (femur), skin, breast, and skeletalmuscle.

Example 4 Production of IFN-L Polypeptides A. Expression of IFN-LPolypeptides in Bacteria

PCR was used to amplify template DNA sequences encoding either human orrat IFN-L polypeptide using primers that corresponded to the 5′ and 3′ends of the sequence (Table I) and which incorporated restriction enzymesites to permit insertion of the amplified product into an expressionvector. Following amplification, PCR products were gel purified,digested with the appropriate restriction enzymes, and ligated into theexpression vector pAMG21 (ATCC No. 98113) using standard recombinant DNAtechniques. After the ligation of PCR insert and vector sequences, theligation reaction mixtures were used to transform an E. coli host strain(e.g., Amgen strain #2596) by electroporation and transformants wereselected for kanamycin drug resistance. Plasmid DNA from selectedcolonies was isolated and subjected to DNA sequencing to confirm thepresence of an appropriate insert.

To construct a rat IFN-L polypeptide bacterial expression vector, IFN-Lnucleic acid sequences were amplified from a cDNA template using theprimers 1825-22 and 1825-21. The PCR product that was obtained followingamplification with these primers was inserted into the Nde I and Bam HIsites of pAMG21, and the ligation reaction was then used in bacterialtransformation. The resulting bacterial clone was designated Amgenstrain #3729. FIG. 4 illustrates the nucleotide sequence of the pAMG21insert of Amgen strain #3729 and the predicted amino acid sequenceencoded by this insert.

A rat IFN-L polypeptide bacterial expression vector, in which thecysteine at position 180 was substituted with a serine residue, wasconstructed using the primers 1825-22 and 1909-56. The PCR product thatwas obtained following amplification with these primers was insertedinto the Nde I and Bam HI sites of pAMG21, and the ligation reaction wasthen used in bacterial transformation.

The resulting bacterial clone was designated Amgen strain #3858. FIG. 5illustrates the nucleotide sequence of the pAMG21 insert of Amgen strain#3858 and the predicted amino acid sequence encoded by this insert.

To construct a human IFN-L polypeptide bacterial expression vector,IFN-L nucleic acid sequences were amplified from a cDNA template usingthe primers 1967-32 and 1982-14. The PCR product that was obtainedfollowing amplification with these primers was inserted into the Xba Iand Bam HI sites of pAMG21, and the ligation reaction was then used inbacterial transformation. The resulting bacterial clone was designatedAmgen strain #4047. FIG. 6 illustrates the nucleotide sequence of thepAMG21 insert of Amgen strain #4047 and the predicted amino acidsequence encoded by this insert.

A human IFN-L polypeptide bacterial expression vector, in which thecysteine at position 193 was substituted with a serine residue, wasconstructed using the primers 1967-32 and 1967-33. The PCR product thatwas obtained following amplification with these primers was insertedinto the Xba I and Bam HI sites of pAMG21, and the ligation reaction wasthen used in bacterial transformation. The resulting bacterial clone wasdesignated Amgen strain #3969. FIG. 7 illustrates the nucleotidesequence of the pAMG21 insert of Amgen strain #3969 and the predictedamino acid sequence encoded by this insert.

A human IFN-L polypeptide bacterial expression vector, expressing anN-terminal variant of human IFN-L polypeptide, was constructed byamplifying plasmid from strain #4047 with the primers 1967-32 and1967-33. The PCR product that was obtained following amplification withthese primers was inserted into the Nde I and Bam HI sites of pAMG21,and the ligation reaction was then used in bacterial transformation. Theresulting bacterial clone was designated Amgen strain #4182. FIG. 8illustrates the nucleotide sequence of the pAMG21 insert of Amgen strain#4182 and the predicted amino acid sequence encoded by this insert.

To generate IFN-L polypeptides, transformed host cells were firstincubated in Terrific Broth medium containing 50 μg/mL kanamycin at 30°C. prior to induction of IFN-L polypeptide. Expression of IFN-Lpolypeptide was induced by the addition of 30 ng/mLN-(3-oxohexanoyl)-dl-homoserine lactone followed by a six hourincubation at either 30° C. or 37° C. Expression of IFN-L polypeptidewas evaluated by centrifugation of the culture, resuspension and lysisof the bacterial pellets, and analysis of host cell proteins bySDS-polyacrylamide gel electrophoresis.

A single band on an SDS polyacrylamide gel corresponding to E. coliproduced IFN-L polypeptide was excised from the gel and N-terminal aminoacid sequence was determined essentially as described by Matsudaira etal., 1987, J. Biol. Chem. 262:10-35).

IFN-L polypeptides were purified as follows. Cells were first lysed inwater by high pressure homogenization and inclusion bodies wereharvested by centrifugation. Solubilized inclusion bodies were thensubjected to a variety of refold conditions.

B. Construction of IFN-L Polypeptide Mammalian Expression Vectors

Native protein and native protein-Fc fusion versions of both human andrat IFN-L polypeptides were produced in either a CHO or 293 mammalianexpression system. Template DNA sequences encoding IFN-L polypeptidewere amplified by PCR using primers corresponding to the 5′ and 3′ ends(Table II).

To construct IFN-L polypeptide expression vectors, IFN-L nucleic acidsequences were amplified as described below. Rat IFN-L nucleic acidsequences were obtained using one of three primer pairs (the forwardprimer 1847-77 and either 1847-88, 1896-56, or 1896-57). A rat IFN-Lpolypeptide-Fc fusion construct was generated by cloning PCR productsprepared with the first set of primers, which incorporated Hind III andNot I cloning sites and no stop codon. Rat IFN-L soluble polypeptideswere generated by cloning PCR products prepared with the second set ofprimers, which incorporated Hind III and Sal I cloning sites and twostop codons, into pDSRα, or the third set of primers, which incorporatedHind III and Not I cloning sites and two stop codons, into pCEP4. HumanIFN-L nucleic acid sequences were obtained using one of three primerpairs (the forward primer 1954-48 and 1954-49 and the forward primer1955-44 and either 1854-45 or 1854-46). A human IFN-L polypeptide-Fcfusion construct was generated by cloning PCR products prepared with thefirst set of primers, which incorporated Not I cloning sites, no stopcodon, and a Factor Xa cleavage site. Human IFN-L soluble polypeptideswere generated by cloning PCR products prepared with the second set ofprimers, which incorporated Hind III and Sal I cloning sites and twostop codons, into pDSRα, or the third set of primers, which incorporatedHind III and Not I cloning sites and two stop codons, into pCEP4. Asecond forward primer (1954-47) was also utilized in place of 1955-44 togenerate constructs possessing two initiation codons.

PCR amplifications were performed using a Perkin-Elmer 9600 thermocyclerand the following reaction conditions: 20 ng of rat or human IFN-Lpolypeptide cDNA, 20 pmol each of the appropriate primers, 1 mmol ofdNTPs, 4 mM MgCl₂, 1×PCR buffer, and SU Taq polymerase (PE Biosystems).Amplification reactions were carried out at 94° C. for 30 seconds, 50°C. for 30 seconds, and 72° C. for 1 minute for 4 cycles followed by 94°C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 1 minute for 26cycles.

PCR products were purified using Qiagen PCR purification spin columnsand then subjected to digestion with the appropriate restrictionendonucleases. Following digestion, fragments were separated on agarosegels, purified using Qiagen gel purification spin columns, and ligatedinto the appropriate vectors. Ligations were transformed into the E.coli strain DH10. Following sequence analysis of selected transformants,large-scale plasmid stocks were prepared for tissue culturetransfection.

C. Expression and Purification of IFN-L Polypeptide in Mammalian Cells

IFN-L polypeptide expression constructs were introduced into 293 EBNA orCHO cells using either a lipofection or calcium phosphate protocol.

To conduct functional studies on the IFN-L polypeptides that wereproduced, large quantities of conditioned media were generated from apool of hygromycin selected 293 EBNA clones. The cells were cultured in500 cm Nunc Triple Flasks to 80% confluence before switching to serumfree media a week prior to harvesting the media. Conditioned media washarvested and frozen at −20° C. until purification.

Conditioned media was purified by affinity chromatography as describedbelow. The media was thawed and then passed through a 0.2 μm filter. AProtein G column was equilibrated with PBS at pH 7.0, and then loadedwith the filtered media. The column was washed with PBS until theabsorbance at A₂₈₀ reached a baseline. IFN-L polypeptide was eluted fromthe column with 0.1 M Glycine-HCl at pH 2.7 and immediately neutralizedwith 1 M Tris-HCl at pH 8.5. Fractions containing IFN-L polypeptide werepooled, dialyzed in PBS, and stored at −70° C.

For Factor Xa cleavage of the human IFN-L polypeptide-Fc fusionpolypeptide, affinity chromatography-purified protein was dialyzed in 50mM Tris-HCl, 100 mM NaCl, 2 mM CaCl₂ at pH 8.0. The restriction proteaseFactor Xa was added to the dialyzed protein at 1/100 (w/w) and thesample digested overnight at room temperature.

Example 5 Biological Activity of IFN-L Polypeptides

The phosphorylation of IFN-L polypeptide was assayed as follows. Celllines were exposed to 1 μg/mL of the rat IFN-L Fc fusion polypeptidegenerated in Example 4C or to a control solution at 37° C. for 15minutes. Following IFN-L polypeptide exposure, the cells were lysed andcellular proteins were recovered and separated by SDS-PAGE. Theseparated proteins were then analyzed by Western blot using an anti-pTyrantibody. Several cell lines showed an increase in cellular proteinphosphorylation following exposure to IFN-L Fc fusion polypeptide.

Example 6 Production of Anti-IFN-L Polypeptide Antibodies

Antibodies to IFN-L polypeptides may be obtained by immunization withpurified protein or with IFN-L peptides produced by biological orchemical synthesis. Suitable procedures for generating antibodiesinclude those described in Hudson and Bay, Practical Immunology (2nded., Blackwell Scientific Publications).

In one procedure for the production of antibodies, animals (typicallymice or rabbits) are injected with an IFN-L antigen (such as an IFN-Lpolypeptide), and those with sufficient serum titer levels as determinedby ELISA are selected for hybridoma production. Spleens of immunizedanimals are collected and prepared as single cell suspensions from whichsplenocytes are recovered. The splenocytes are fused to mouse myelomacells (such as Sp2/0-Ag14 cells), are first incubated in DMEM with 200U/mL penicillin, 200 μg/mL streptomycin sulfate, and 4 mM glutamine, andare then incubated in HAT selection medium (hypoxanthine, aminopterin,and thymidine). After selection, the tissue culture supernatants aretaken from each fusion well and tested for anti-IFN-L antibodyproduction by ELISA.

Alternative procedures for obtaining anti-IFN-L antibodies may also beemployed, such as the immunization of transgenic mice harboring human Igloci for production of human antibodies, and the screening of syntheticantibody libraries, such as those generated by mutagenesis of anantibody variable domain.

Example 7 Expression of IFN-L Polypeptide in Transgenic Mice

To assess the biological activity of IFN-L polypeptide, a constructencoding an IFN-L polypeptide/Fc fusion protein under the control of aliver specific ApoE promoter is prepared. The delivery of this constructis expected to cause pathological changes that are informative as to thefunction of IFN-L polypeptide. Similarly, a construct containing thefull-length IFN-L polypeptide under the control of the beta actinpromoter is prepared. The delivery of this construct is expected toresult in ubiquitous expression.

To generate these constructs, PCR is used to amplify template DNAsequences encoding an IFN-L polypeptide using primers that correspond tothe 5′ and 3′ ends of the desired sequence and which incorporaterestriction enzyme sites to permit insertion of the amplified productinto an expression vector. Following amplification, PCR products are gelpurified, digested with the appropriate restriction enzymes, and ligatedinto an expression vector using standard recombinant DNA techniques. Forexample, amplified IFN-L polypeptide sequences can be cloned into anexpression vector under the control of the human β-actin promoter asdescribed by Graham et al., 1997, Nature Genetics, 17:272-74 and Ray etal., 1991, Genes Dev. 5:2265-73.

Following ligation, reaction mixtures are used to transform an E. colihost strain by electroporation and transformants are selected for drugresistance.

Plasmid DNA from selected colonies is isolated and subjected to DNAsequencing to confirm the presence of an appropriate insert and absenceof mutation. The IFN-L polypeptide expression vector is purified throughtwo rounds of CsCl density gradient centrifugation, cleaved with asuitable restriction enzyme, and the linearized fragment containing theIFN-L polypeptide transgene is purified by gel electrophoresis. Thepurified fragment is resuspended in 5 mM Tris, pH 7.4, and 0.2 mM EDTAat a concentration of 2 mg/mL.

Single-cell embryos from BDF1×BDF1 bred mice are injected as described(PCT Pub. No. WO 97/23614). Embryos are cultured overnight in a CO₂incubator and 15-20 two-cell embryos are transferred to the oviducts ofa pseudopregnant CD1 female mice. Offspring obtained from theimplantation of microinjected embryos are screened by PCR amplificationof the integrated transgene in genomic DNA samples as follows. Earpieces are digested in 20 mL ear buffer (20 mM Tris, pH 8.0, 10 mM EDTA,0.5% SDS, and 500 mg/mL proteinase K) at 55° C. overnight. The sample isthen diluted with 200 mL of TE, and 2 mL of the ear sample is used in aPCR reaction using appropriate primers.

At 8 weeks of age, transgenic founder animals and control animals aresacrificed for necropsy and pathological analysis. Portions of spleenare removed and total cellular RNA isolated from the spleens using theTotal RNA Extraction Kit (Qiagen) and transgene expression determined byRT-PCR. RNA recovered from spleens is converted to cDNA using theSuperScript™ Preamplification System (Gibco-BRL) as follows. A suitableprimer, located in the expression vector sequence and 3′ to the IFN-Lpolypeptide transgene, is used to prime cDNA synthesis from thetransgene transcripts. Ten mg of total spleen RNA from transgenicfounders and controls is incubated with 1 mM of primer for 10 minutes at70° C. and placed on ice. The reaction is then supplemented with 10 mMTris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl₂, 10 mM of each dNTP, 0.1 mMDTT, and 200 U of SuperScript II reverse transcriptase. Followingincubation for 50 minutes at 42° C., the reaction is stopped by heatingfor 15 minutes at 72° C. and digested with 2 U of RNase H for 20 minutesat 37° C. Samples are then amplified by PCR using primers specific forIFN-L polypeptide.

Example 8 Biological Activity of IFN-L Polypeptide in Transgenic Mice

Prior to euthanasia, transgenic animals are weighed, anesthetized byisofluorane and blood drawn by cardiac puncture. The samples aresubjected to hematology and serum chemistry analysis. Radiography isperformed after terminal exsanguination. Upon gross dissection, majorvisceral organs are subject to weight analysis.

Following gross dissection, tissues (i.e., liver, spleen, pancreas,stomach, the entire gastrointestinal tract, kidney, reproductive organs,skin and mammary glands, bone, brain, heart, lung, thymus, trachea,esophagus, thyroid, adrenals, urinary bladder, lymph nodes and skeletalmuscle) are removed and fixed in 10% buffered Zn-Formalin forhistological examination. After fixation, the tissues are processed intoparaffin blocks, and 3 mm sections are obtained. All sections arestained with hematoxylin and exosin, and are then subjected tohistological analysis.

The spleen, lymph node, and Peyer's patches of both the transgenic andthe control mice are subjected to immunohistology analysis with B celland T cell specific antibodies as follows. The formalin fixed paraffinembedded sections are deparaffinized and hydrated in deionized water.The sections are quenched with 3% hydrogen peroxide, blocked withProtein Block (Lipshaw, Pittsburgh, Pa.), and incubated in ratmonoclonal anti-mouse B220 and CD3 (Harlan, Indianapolis, Ind.).Antibody binding is detected by biotinylated rabbit anti-ratimmunoglobulins and peroxidase conjugated streptavidin (BioGenex, SanRamon, Calif.) with DAB as a chromagen (BioTek, Santa Barbara, Calif.).Sections are counterstained with hematoxylin.

After necropsy, MLN and sections of spleen and thymus from transgenicanimals and control littermates are removed. Single cell suspensions areprepared by gently grinding the tissues with the flat end of a syringeagainst the bottom of a 100 mm nylon cell strainer (Becton Dickinson,Franklin Lakes, N.J.). Cells are washed twice, counted, andapproximately 1×10⁶ cells from each tissue are then incubated for 10minutes with 0.5 μg CD16/32(FcγIII/II) Fc block in a 20 μL volume.Samples are then stained for 30 minutes at 2-8° C. in a 100 μL volume ofPBS (lacking Ca⁺ and Mg⁺), 0.1% bovine serum albumin, and 0.01% sodiumazide with 0.5 μg antibody of FITC or PE-conjugated monoclonalantibodies against CD90.2 (Thy-1.2), CD45R (B220), CD11b(Mac-1), Gr-1,CD4, or CD8 (PharMingen, San Diego, Calif.). Following antibody binding,the cells are washed and then analyzed by flow cytometry on a FACScan(Becton Dickinson).

While the present invention has been described in terms of the preferredembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations that come withinthe scope of the invention as claimed.

1. An antibody that specifically binds the polypeptide comprising theamino acid sequence as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5,or a fragment thereof.
 2. The antibody of claim 1 that is a humanizedantibody.
 3. The antibody of claim 1 that is a human antibody orfragment thereof.
 4. The antibody of claim 1 that is a polyclonalantibody or fragment thereof.
 5. The antibody of claim 1 that is amonoclonal antibody or fragment thereof.
 6. The antibody of claim 1 thatis a chimeric antibody or fragment thereof.
 7. The antibody of claim 1that is a CDR-grafted antibody or fragment thereof.
 8. The antibody ofclaim 1 that is a variable region fragment.
 9. The variable regionfragment of claim 1 that is a Fab or a Fab′ fragment.
 10. The antibodyof claim 1 that is bound to a detectable label.
 11. A antibody producedby immunizing an animal with a polypeptide comprising an amino acidsequence of either SEQ ID NO: 2 or SEQ ID NO:
 5. 12. A hybridoma whichproduces an antibody which is capable of binding a polypeptide accordingto any of claim 1.